Methods of decreasing ZVEGF3 activity

ABSTRACT

Polypeptide growth factors, methods of making them, polynucleotides encoding them, antibodies to them, and methods of using them are disclosed. The polypeptides comprise an amino acid segment that is at least 90% identical to residues 46-163 of SEQ ID NO: 2 or residues 235-345 of SEQ ID NO: 2. Multimers of the polypeptides are also disclosed. The polypeptides, multimeric proteins, and polynucleotides can be used in the study and regulation of cell and tissue development, as components of cell culture media, and as diagnostic agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional application of co-pending application Ser. No.09/457,066, filed Dec. 7, 1999, which claims benefit under 35 U.S.C. §119(e) of provisional applications No. 60/111,173, filed Dec. 7, 1998;Ser. No. 60/142,576 filed Jul. 6, 1999; Ser. No. 60/161,653 filed Oct.21, 1999 and Ser. No. 60/165,255 filed Nov. 12, 1999.

BACKGROUND OF THE INVENTION

In multicellular animals, cell growth, differentiation, and migrationare controlled by polypeptide growth factors. These growth factors playa role in both normal development and pathogenesis, including thedevelopment of solid tumors.

Polypeptide growth factors influence cellular events by binding tocell-surface receptors, many of which are tyrosine kinases. Bindinginitiates a chain of signalling events within the cell, which ultimatelyresults in phenotypic changes, such as cell division, proteaseproduction, and cell migration.

Growth factors can be classified into families on the basis ofstructural similarities. One such family, the PDGF (platelet derivedgrowth factor) family, is characterized by a dimeric structurestabilized by disulfide bonds. This family includes PDGF, the placentalgrowth factors (PlGFs), and the vascular endothelial growth factors(VEGFs). The individual polypeptide chains of these proteins formcharacteristic higher-order structures having a bow tie-likeconfiguration about a cystine knot, formed by disulfide bonding betweenpairs of cysteine residues. Hydrophobic interactions between loopscontribute to the dimerization of the two monomers. See, Daopin et al.,Science 257:369, 1992; Lapthorn et al., Nature 369:455, 1994. Members ofthis family are active as both homodimers and heterodimers. See, forexample, Heldin et al., EMBO J. 7:1387-1393, 1988; Cao et al., J. Biol.Chem. 271:3154-3162, 1996. The cystine knot motif and bow tie fold arealso characteristic of the growth factors transforming growthfactor-beta (TGF-β) and nerve growth factor (NGF), and the glycoproteinhormones. Although their amino acid sequences are quite divergent, theseproteins all contain the six conserved cysteine residues of the cystineknot.

Four vascular endothelial growth factors have been identified: VEGF,also known as vascular permeability factor (Dvorak et al., Am. J.Pathol. 146:1029-1039, 1995); VEGF-B (Olofsson et al., Proc. Natl. Acad.Sci. USA 93:2567-2581, 1996; Hayward et al., WIPO Publication WO96/27007); VEGF-C (Joukov et al., EMBO J. 15:290-298, 1996); and VEGF-D(Oliviero, WO 97/12972; Achen et al., WO 98/07832). Five VEGFpolypeptides (121, 145, 165, 189, and 206 amino acids) arise fromalternative splicing of the VEGF mRNA.

VEGFs stimulate the development of vasculature through a process knownas angiogenesis, wherein vascular endothelial cells re-enter the cellcycle, degrade underlying basement membrane, and migrate to form newcapillary sprouts. These cells then differentiate, and mature vesselsare formed. This process of growth and differentiation is regulated by abalance of pro-angiogenic and anti-angiogenic factors. Angiogenesis iscentral to normal formation and repair of tissue, occuring in embryodevelopment and wound healing. Angiogenesis is also a factor in thedevelopment of certain diseases, including solid tumors, rheumatoidarthritis, diabetic retinopathy, macular degeneration, andatherosclerosis.

A number of proteins from vertebrates and invertebrates have beenidentified as influencing neural development. Among those molecules aremembers of the neuropilin family and the semaphorin/collapsin family.Neuronal cell outgrowths, known as processes, grow away from the cellbody to form synaptic connections. Long, thin processes that carryinformation away from the cell body are called axons, and short, thickerprocesses which carry information to and from the cell body are calleddendrites. Axons and dendrites are collectively referred to as neurites.Neurites are extended by means of growth cones, the growing tips ofneurites, which are highly motile and are ultimately responsible forincreasing and extending the neuronal network in the body.

Three receptors for VEGF have been identified: KDR/Flk-1 (Matthews etal., Proc. Natl. Acad. Sci. USA 88:9026-9030, 1991), Flt-1 (de Vries etal., Science 255:989-991, 1992), and neuropilin-1 (Soker et al., Cell92:735-745, 1998). Neuropilin-1 is a cell-surface glycoprotein that wasinitially identified in Xenopus tadpole nervous tissues, then inchicken, mouse, and human. The primary structure of neuropilin-1 ishighly conserved among these vertebrate species. Neuropilin-1 has beendemonstrated to be a receptor for various members of the semaphorinfamily including semaphorin III (Kolodkin et al., Cell 90:753-762,1997), Sema E and Sema IV (Chen et al., Neuron 19:547-559, 1997). Avariety of activities have been associated with the binding ofneuropilin-1 to its ligands. For example, binding of semaphorin III toneuropilin-1 can induce neuronal growth cone collapse and repulsion ofneurites in vitro (Kitsukawa et al., Neuron 19: 995-1005, 1997).

In mice, neuropilin-1 is expressed in the cardiovascular system, nervoussystem, and limbs at particular developmental stages. Chimeric miceover-expressing neuropilin-1 were found to be embryonic lethal(Kitsukawa et al., Development 121:4309-4318, 1995). The chimericembryos exhibited several morphological abnormalities, including excesscapillaries and blood vessels, dilation of blood vessels, malformedhearts, ectopic sprouting and defasciculation of nerve fibers, and extradigits. All of these abnormalities occurred in the organs in whichneuropilin-1 is expressed in normal development. Mice lacking theneuropilin-1 gene have severe cardiovascular abnormalities, includingimpairment of vascular network formation in the central and peripheralnervous systems (Takashima et al., American Heart Association 1998Meeting, Abstract # 3178).

Neuropilin-1 has been identified as a cell-surface receptor for VEGF(Soker et al., ibid.), and displays selective binding activity forVEGF₁₆₅ over VEGF₁₂₁. It has been shown to be expressed on vascularendothelial cells and tumor cells in vitro. When neuropilin-1 isco-expressed in cells with KDR, neuropilin-1 enhances the binding ofVEGF₁₆₅ to KDR and VEGF₁₆₅-mediated chemotaxis. Conversely, inhibitionof VEGF₁₆₅ binding to neuropilin-1 inhibits its binding to KDR and itsmitogenic activity for endothelial cells (Soker, et al., ibid.).Neuropilin-1 is also a receptor for PlGF-2 (Migdal et al., J. Biol.Chem. 273: 22272-22278, 1998). A second semaphorin receptor,neuropilin-2, exhibits homology with neuropilin-1 but has differs inbinding specificity (Chen et al. Neuron 19: 547-559, 1997).

Semaphorins are a large family of molecules which share the definingsemaphorin domain of approximately 500 amino acids. This family can besubdivided into multiple subfamilies that contain both secreted andmembrane-bound proteins. Select members of these subfamilies, class III(SemD) and class IV (SemD), form homodimers linked by disulfide bridges.In the case of SemD, there is additional proteolytic processing thatcreates a 65-kDa isoform that lacks the 33-kDa carboxyl-terminalsequence. Dimerization is believed to be important for functionalactivity (Klostermann et al., J. Biol. Chem. 273:7326-7331, 1998).Collapsin-1, the first identified vertebrate member of the semaphorinfamily of axon guidance proteins, has also been shown to form covalentdimers, with dimerization necessary for collapse activity (Koppel etal., J. Biol. Chem. 273:15708-15713, 1998).

Semaphorin III has been associated in vitro with regulating growth clonecollapse and chemorepulsion of neurites. In vivo it has also been shownto be required for correct sensory afferent innervation and otheraspects of development, including skeletal and cardiac defects (Fehar etal., Nature 383:525-528, 1996). Other members of the semaphorin familyhave been shown to be associated with other forms of biology. The humansemaphorin E gene is expressed in rheumatoid synovial cells and isthought to play an immunosuppressive role via inhibition of cytokines(Mangasser-Stephan et al., Biochem. Biophys. Res. Comm. 234:153-156,1997). CD100, a leukocyte semaphorin, promotes B-cell aggregation anddifferentiation (Hall et al., Proc. Natl. Acad. Sci. USA 93:11780-11785,1996). CD100 has also been shown to be expressed in many T-celllymphomas and may be a marker of malignant T-cell neoplasms (Dorfman etal., Am. J. Pathol. 153:255-262, 1998). Semaphorin homologues have alsobeen identified in DNA viruses (Lang, Genomics 51:340-350, 1998) and inpoxvirus (Comeau, et al. Immunity 8:473-482, 1998). Transcription of themouse semaphorin gene, M-semaH, correlates with metastatic ability ofmouse tumor cell lines (Christensen et al., Cancer Res. 58:1238-1244,1998).

The role of growth factors, other regulatory molecules, and theirreceptors in controlling cellular processes makes them likely candidatesand targets for therapeutic intervention. Platelet-derived growthfactor, for example, has been disclosed for the treatment of periodontaldisease (U.S. Pat. No. 5,124,316), gastrointestinal ulcers (U.S. Pat.No. 5,234,908), and dermal ulcers (Robson et al., Lancet 339:23-25,1992; Steed et al., J. Vasc. Surg. 21:71-81, 1995). Inhibition of PDGFreceptor activity has been shown to reduce intimal hyperplasia ininjured baboon arteries (Giese et al., Restenosis Summit VIII, PosterSession #23, 1996; U.S. Pat. No. 5,620,687). Vascular endothelial growthfactors (VEGFs) have been shown to promote the growth of blood vesselsin ischemic limbs (Isner et al., The Lancet 348:370-374, 1996), and havebeen proposed for use as wound-healing agents, for treatment ofperiodontal disease, for promoting endothelialization in vascular graftsurgery, and for promoting collateral circulation following myocardialinfarction (WIPO Publication No. WO 95/24473; U.S. Pat. No. 5,219,739).VEGFs are also useful for promoting the growth of vascular endothelialcells in culture. A soluble VEGF receptor (soluble flt-1) has been foundto block binding of VEGF to cell-surface receptors and to inhibit thegrowth of vascular tissue in vitro (Biotechnology News 16(17):5-6,1996).

In view of the proven clinical utility of hormones, there are needs inthe art for additional such molecules for use as therapeutic agents,diagnostic. agents, and research tools and reagents. These and otherneeds are addressed by the present invention.

DESCRIPTION OF THE INVENTION

The present invention provides an isolated polypeptide of at least 15amino acid residues comprising an epitope-bearing portion of a proteinof SEQ ID NO:2. Within certain embodiments, the polypeptide comprises asegment that is at least 90% identical to residues 46-163 of SEQ ID NO:2or residues 235-345 of SEQ ID NO:2. Within additional embodiments, thepolypeptide is selected from the group consisting of residues 15-163 ofSEQ ID NO:2, residues 46-163 of SEQ ID NO:2, residues 15-170 of SEQ IDNO:2, residues 46-170 of SEQ ID NO:2, residues 15-234 of SEQ ID NO:2,residues 46-234 of SEQ ID NO:2, residues 15-229 amide of SEQ ID NO:2,residues 15-230 of SEQ ID NO:2, residues 15-345 of SEQ ID NO:2, residues46-345 of SEQ ID NO:2, residues 235-345 of SEQ ID NO:2, and residues226-345 of SEQ ID NO:2.

The invention also provides an isolated polypeptide comprising asequence of amino acids of the formula R1_(x)-R2_(y)-R3_(z), wherein R1comprises a polypeptide of from 100 to 120 residues in length that is atleast 90% identical to residues 46-163 of SEQ ID NO:2, and comprises asequence motif C[KR]Y[DNE] [WYF]X{{11,15}}G[KR] [WYF]C (SEQ ID NO:4)corresponding to residues 104-124 of SEQ ID NO:2; R2 is a polypeptide atleast 90% identical to residues 164-234 of SEQ ID NO:2; R3 is apolypeptide at least 90% identical in amino acid sequence to residues235-345 of SEQ ID NO:2 and comprises cysteine residues at positionscorresponding to residues 250, 280, 284, 296, 335, and 337 of SEQ IDNO:2, a glycine residue at a position corresponding to residue 282 ofSEQ ID NO:2, and a sequence motif CX{18,33}CXGXCX{6,33}CX{20,40}CXC (SEQID NO:3) corresponding to residues 250-337 of SEQ ID NO:2; and each ofx, y, and z is individually 0 or 1, subject to the limitations that atleast one of x and z is 1, and, if x and z are each 1, then y is 1.There are thus provided isolated polypeptides of the above formulawherein (a) x=1, (b) z=1, and (c) x=1 and z=1. Within certainembodiments, x=1 and R1 is at least 90% identical to residues 18-163 ofSEQ ID NO:2. Within related embodiments, x=1 and R1 comprises residues46-163 of SEQ ID NO:2. Within other embodiments, z=1 and R3 comprisesresidues 235-345 of SEQ ID NO:2. Within additional embodiments, x=1,z=1, and the polypeptide comprises residues 46-229 of SEQ ID NO:2,residues 164-345 of SEQ ID NO:2, or residues 46-345 of SEQ ID NO:2. Theisolated polypeptide may further comprise cysteine residues at positionscorresponding to residues 286, 287, 291, and 294 of SEQ ID NO:2. Withinother embodiments, the isolated polypeptide further comprises anaffinity tag. Within a related embodiment, the isolated polypeptidecomprises an immunoglobulin constant domain.

The present invention also provides an isolated protein comprising afirst polypeptide operably linked to a second polypeptide, wherein thefirst polypeptide comprises a sequence of amino acids of the formulaR1_(x)-R2_(y)-R3_(z) as disclosed above. The protein modulates cellproliferation, differentiation, metabolism, or migration. Within oneembodiment, the protein is a heterodimer. Within related embodiments,the second polypeptide is selected from the group consisting of VEGF,VEGF-B, VEGF-C, VEGF-D, zvegf4, PlGF, PDGF-A, and PDGF-B. Within anotherembodiment, the protein is a homodimer.

There is also provided an isolated protein produced by a methodcomprising the steps of (a) culturing a host cell containing anexpression vector comprising the following operably linked elements: atranscription promoter; a DNA segment encoding a polypeptide selectedfrom the group consisting of (i) residues 46-345 of SEQ ID NO:2, (ii)residues 46-234 of SEQ ID NO:2, (iii) residues 164-345 of SEQ ID NO:2,and (iv) residues 235-345 of SEQ ID NO:2; and a transcriptionterminator, under conditions whereby the DNA segment is expressed; and(b) recovering from the cell the protein product of expression of theDNA construct.

Within another aspect of the invention there is provided an isolatedpolynucleotide of up to approximately 4 kb in length, wherein saidpolynucleotide encodes a polypeptide as disclosed above. Within oneembodiment of the invention, the polynucleotide is DNA.

Within a further aspect of the invention there is provided an expressionvector comprising the following operably linked elements: (a) atranscription promoter; (b) a DNA polynucleotide as disclosed above; and(c) a transcription terminator. The vector may further comprise asecretory signal sequence operably linked to the DNA polynucleotide.

Also provided by the invention is a cultured cell into which has beenintroduced an expression vector as disclosed above, wherein the cellexpresses the polypeptide encoded by the DNA segment. The cultured cellcan be used within a method of producing a polypeptide, the methodcomprising culturing the cell and recovering the expressed polypeptide.

The proteins provided herein can be combined with a pharmaceuticallyacceptable vehicle to provide a pharmaceutical composition.

The invention also provides an antibody that specifically binds to anepitope of a polypeptide as disclosed above. Antibodies of the inventioninclude, inter alia, monoclonal antibodies and single chain antibodies,and may be linked to a reporter molecule.

The invention further provides a method for detecting a geneticabnormality in a patient, comprising the steps of (a) obtaining agenetic sample from a patient, (b) incubating the genetic sample with apolynucleotide comprising at least 14 contiguous nucleotides of SEQ IDNO:1 or the complement of SEQ ID NO:1, under conditions wherein saidpolynucleotide will hybridize to complementary polynucleotide sequence,to produce a first reaction product, and (c) comparing the firstreaction product to a control reaction product, wherein a differencebetween the first reaction product and the control reaction product isindicative of a genetic abnormality in the patient.

Within an additional aspect, the invention provides a method ofstimulating the growth of fibroblasts or smooth muscle cells comprisingapplying to the cells an effective amount of a protein as disclosedabove.

Within another aspect the invention provides methods for modulating cellgrowth or other cellular processes. Within one embodiment there isprovided a method of stimulating the growth of fibroblasts or smoothmuscle cells comprising applying to the cells an effective amount of aprotein as disclosed above. Within another embodiment there is provideda method of activating a cell-surface PDGF alpha receptor, comprisingexposing a cell comprising a cell-surface PDGF alpha receptor to apolypeptide or protein as disclosed above, whereby the polypeptide orprotein binds to and activates the receptor. Within a further embodimentthere is provided a method of inhibiting a PDGF alpha receptor-mediatedcellular process, comprising exposing a cell comprising a cell-surfacePDGF alpha receptor to a compound that inhibits binding of a polypeptideor protein as disclosed above to the receptor.

Within a further method of the invention there is provided a method ofinhibiting zvegf3 activity in a mammal comprising administering to themammal an effective amount of a zvegf3 antagonist. Within certainembodiments the antagonist is an antibody, a receptor, a ligand-bindingreceptor fragment, or a receptor IgG-Fc fusion protein.

Within another aspect of the invention there is provided an isolated,antisense polynucleotide that is the complement of a polynucleotideencoding a polypeptide comprising a sequence of amino acids of theformula R1_(x)-R2_(y)-R3_(z), wherein R1 comprises a polypeptide of from100 to 120 residues in length that is at least 90% identical to residues46-163 of SEQ ID NO:2, and comprises a sequence motif C[KR]Y[DNE][WYF]X{11,15}G[KR] [WYF]C (SEQ ID NO:4) corresponding to residues104-124 of SEQ ID NO:2; R2 is a polypeptide at least 90% identical toresidues 164-234 of SEQ ID NO:2; R3 is a polypeptide at least 90%identical in amino acid sequence to residues 235-345 of SEQ ID NO:2 andcomprises cysteine residues at positions corresponding to residues 250,280, 284, 296, 335, and 337 of SEQ ID NO:2, a glycine residue at aposition corresponding to residue 282 of SEQ ID NO:2, and a sequencemotif CX{18,33}CXGXCX{6,33}CX{20,40}CXC (SEQ ID NO:3) corresponding toresidues 250-337 of SEQ ID NO:2; and each of x, y, and z is individually0 or 1, subject to the limitations that at least one of x and z is 1,and, if x and z are each 1, then y is 1. Within one embodiment theantisense polynucleotide further comprises operably linked transcriptionpromoter and terminator sequences. The antisense polynucleotide can beused within a method of inhibiting zvegf3 production in a cellcomprising administering to the cell the antisense polynucleotide.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention and theattached drawings. In the drawings:

FIG. 1 is a Hopp/Woods hydrophilicity profile of the amino acid sequenceshown in SEQ ID NO:2. The profile is based on a sliding six-residuewindow. Buried G, S, and T residues and exposed H, Y, and W residueswere ignored. These residues are indicated in the figure by lower caseletters.

FIG. 2 is an illustration of the vector pHB12-8 for use in expressingcDNAs in transgenic animals.

FIG. 3 is an illustration of the vector pZMP6/zvegf3.

FIG. 4 is an illustration of the vector pZMP11/zv3GF-otPA.

FIG. 5 shows the results of a receptor binding assay for zvegf3.

FIG. 6 is an alignment of human (SEQ ID NO:2) and mouse (SEQ ID NO:43)amino acid sequences.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), maltose binding protein(Kellerman and Ferenci, Methods Enzymol. 90:459-463, 1982; Guan et al.,Gene 67:21-30, 1987), Glu-Glu affinity tag (Grussenmeyer et al., Proc.Natl. Acad. Sci. USA 82:7952-4, 1985; see SEQ ID NO:5), substance P,Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988), streptavidinbinding peptide, thioredoxin, ubiquitin, cellulose binding protein, T7polymerase, or other antigenic epitope or binding domain. See, ingeneral, Ford et al., Protein Expression and Purification 2: 95-107,1991. DNAs encoding affinity tags and other reagents are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; NewEngland Biolabs, Beverly, Mass.; and Eastman Kodak, New Haven, Conn.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal”, and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

A “beta-strand-like region” is a region of a protein characterized bycertain combinations of the polypeptide backbone dihedral angles phi (φ)and psi (ψ). Regions wherein φ is less than −60° and ψ is greater than90° are beta-strand-like. Those skilled in the art will recognize thatthe limits of a β-strand are somewhat imprecise and may vary with thecriteria used to define them. See, for example, Richardson andRichardson in Fasman, ed., Prediction of Protein Structure and thePrinciples of Protein Conformation, Plenum Press, New York, 1989; andLesk, Protein Architecture: A Practical Approach, Oxford UniversityPress, New York, 1991.

A “complement” of a polynucleotide molecule is a polynucleotide moleculehaving a complementary base sequence and reverse orientation as comparedto a reference sequence. For example, the sequence 5′ ATGCACGGG 3′ iscomplementary to 5′ CCCGTGCAT 3′.

“Corresponding to”, when used in reference to a nucleotide or amino acidsequence, indicates the position in a second sequence that aligns withthe reference position when two sequences are optimally aligned.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide)Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. Within one form, the isolated polypeptideor protein is substantially free of other polypeptides or proteins,particularly other polypeptides or proteins of animal origin.Polypeptides and proteins can be provided in a highly purified form,i.e. greater than 95% pure or greater than 99% pure. When used in thiscontext, the term “isolated” does not exclude the presence of the samepolypeptide or protein in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

A “motif” is a series of amino acid positions in a protein sequence forwhich certain amino acid residues are required. A motif defines the setof possible residues at each such position.

“Operably linked” means that two or more entities are joined togethersuch that they function in concert for their intended purposes. Whenreferring to DNA segments, the phrase indicates, for example, thatcoding sequences are joined in the correct reading frame, andtranscription initiates in the promoter and proceeds through the codingsegment(s) to the terminator. When referring to polypeptides, “operablylinked” includes both covalently (e.g., by disulfide bonding) andnon-covalently (e.g., by hydrogen bonding, hydrophobic interactions, orsalt-bridge interactions) linked sequences, wherein the desiredfunction(s) of the sequences are retained.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

A “PDGF alpha receptor-mediated cellular process” is a cellular processthat occurs in response to activation of a PDGF alpha receptor. Suchprocesses include, without limitation, cell division, chemotaxis, celldifferentiation, and production or release of macromolecules.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired. Such unpaired ends will ingeneral not exceed 20 nt in length.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A “secretory signal sequence” is a DNA sequence that encodes apolypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

A “segment” is a portion of a larger molecule (e.g., polynucleotide orpolypeptide) having specified attributes. For example, a DNA segmentencoding a specified polypeptide is a portion of a longer DNA molecule,such as a plasmid or plasmid fragment, that, when read from the 5′ tothe 3′ direction, encodes the sequence of amino acids of the specifiedpolypeptide.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±20%.

All references cited herein are incorporated by reference in theirentirety.

The present invention is based in part upon the discovery of a novel DNAmolecule that encodes a polypeptide comprising a growth factor domainand a CUB domain. The growth factor domain is characterized by anarrangement of cysteine residues and beta strands that is characteristicof the “cystine knot” structure of the PDGF family. The CUB domain showssequence homology to CUB domains in the neuropilins (Takagi et al.,Neuron 7:295-307, 1991; Soker et al., ibid.), human bone morphogeneticprotein-1 (Wozney et al., Science 242:1528-1534, 1988), porcine seminalplasma protein and bovine acidic seminal fluid protein (Romero et al.,Nat. Struct. Biol. 4:783-788, 1997), and X. laevis tolloid-like protein(Lin et al., Dev. Growth Differ. 39:43-51, 1997). Analysis of the tissuedistribution of the mRNA corresponding to this novel DNA showed thatexpression was widespread in adult human tissues, and that expressionoccured up to day 15 in mouse embryo. The polypeptide has beendesignated “zvegf3” in view of its homology to the VEGFs in the growthfactor domain.

Structural predictions based on the zvegf3 sequence and its homology toother growth factors suggests that the polypeptide can formhomomultimers or heteromultimers that act on tissues to control organdevelopment by modulating cell proliferation, migration,differentiation, or metabolism. Zvegf3 heteromultimers may comprise apolypeptide from another member of the PDGF/VEGF family of proteins,including VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf4 (SEQ ID NOS: 36 and 37),PlGF (Maglione et al., Proc. Natl. Acad. Sci. USA 88:9267-9271, 1991),PDGF-A (Murray et al., U.S. Pat. No. 4,899,919; Heldin et al., U.S. Pat.No. 5,219,759), or PDGF-B (Chiu et al., Cell 37:123-129, 1984; Johnssonet al., EMBO J. 3:921-928, 1984). Members of this family of polypeptidesregulate organ development and regeneration, post-developmental organgrowth, and organ maintenance, as well as tissue maintenance and repairprocesses. These factors are also involved in pathological processeswhere therapeutic treatments are required, including cancer, rheumatoidarthritis, diabetic retinopathy, ischemic limb disease, peripheralvascular disease, myocardial ischemia, vascular intimal hyperplasia,atherosclerosis, and hemangioma formation. To treat these pathologicalconditions it will often be required to develop compounds to antagonizethe members of the PDGF/VEGF family of proteins, or their respectivereceptors. This may include the development of neutralizing antibodies,small molecule antagonists, modified forms of the growth factors thatmaintain receptor binding activity but lack receptor activatingactivity, soluble receptors, or antisense or ribozyme molecules to blockpolypeptide production.

SEQ ID NO:2 is the sequence of a representative polypeptide of thepresent invention. Analysis of the amino acid sequence shown in SEQ IDNO:2 indicates that residues 1 to 14 form a secretory peptide. The CUBdomain extends from residue 46 to residue 163. A propeptide-likesequence extends from residue 164 to residue 234, and includes twopotential cleavage sites at its carboxyl terminus, a dibasic site atresidues 231-232 and a target site for furin or a furin-like protease atresidues 231-234. The growth factor domain extends from residue 235 toresidue 345. Those skilled in the art will recognize that domainboundaries are somewhat imprecise and can be expected to vary by up to±5 residues from the specified positions. Potential proteolytic cleavagesites occur at residues 232 and 234. Processing of recombinant zvegf3produced in BHK cells has been found to occur between residues 225 and226. Signal peptide cleavage is predicted to occur after residue 14 (±3residues). This analysis suggests that the zvegf3 polypeptide chain maybe cleaved to produce a plurality of monomeric species as shown inTable 1. Cleavage after Arg-234 is expected to result in subsequentremoval of residues 231-234, with possible conversion of Gly-230 to anamide. Cleavage after Lys-232 is expected to result in subsequentremoval of residue 231, again with possible conversion of Gly-230 to anamide. In addition, it may be advantageous to include up to sevenresidues of the interdomain region at the carboxyl terminus of the CUBdomain. The interdomain region can be truncated at its amino terminus bya like amount. See Table 1.

TABLE 1 Monomer Residues (SEQ ID NO:2) Cub domain  15-163  46-163 15-170  46-170 CUB domain + interdomain  15-234 region  46-234  15-229amide  15-230 Cub domain + interdomain  15-345 region + growth factor 46-345 domain Growth factor domain 235-345 226-345 Growth factordomain + 164-345 interdomain region 171-345

Also included within the present invention are polypeptides that are atleast 90% identical or at least 95% identical to the polypeptidesdisclosed in Table 1, wherein these additional polypeptides retaincertain characteristic sequence motifs as disclosed below.

Zvegf3 polypeptides are designated herein with a subscript indicatingthe amino acid residues. For example, the CUB domain polypeptidesdisclosed in Table 1 are designated “zvegf3₁₅₋₁₆₃”, “zvegf3₄₆₋₁₆₃”,“zvegf3₁₅₋₁₇₀”, and “zvegf3₄₆₋₁₇₀”.

Higher order structure of zvegf3 polypeptides can be predicted bysequence alignment with known homologs and computer analysis usingavailable software (e.g., the Insight II® viewer and homology modelingtools; MSI, San Diego, Calif.). Analysis of SEQ ID NO:2 predicts thatthe secondary structure of the growth factor domain is dominated by thecystine knot, which ties together variable beta strand-like regions andloops into a bow tie-like structure. Sequence alignment indicates thatCys residues within the growth factor domain at positions 250, 280, 284,296, 335, and 337, and Gly 282 are highly conserved within the family.Further analysis suggests pairing (disulfide bond formation) of Cysresidues 250 and 296, 280 and 335, and 284 and 337 to form the cystineknot. This arrangement of conserved residues can be represented by theformula CX{18,33}CXGXCX{6,33}CX{20,40}CXC (SEQ ID NO:3), wherein aminoacid residues are represented by the conventional single-letter code, Xis any amino acid residue, and {y,z} indicates a region of variableresidues (X) from y to z residues in length. A consensus bow tiestructure is formed as: amino terminus to cystine knot→beta strand-likeregion 1→variable loop 1→beta strand-like region 2→cystine knot→betastrand-like region 3→variable loop 2→beta strand-like region 4→cystineknot→beta strand-like region 5→variable loop 3→beta strand-like region6→cystine knot. Variable loops 1 and 2 form one side of the bow tie,with variable loop 3 forming the other side. The structure of the zvegf3growth factor domain appears to diverge from the consensus structure ofother family members in loop 2 and beta strand-like regions 3 and 4,wherein all are abbreviated and essentially replaced by a cysteinecluster comprising residues 285 (Ala) through 295 (Gln), which includesCys residues at positions 286, 287, 291, and 294 of SEQ ID NO:2. Theapproximate boundaries of the beta strand-like regions in SEQ ID NO:2are: region 1, residues 251-259; region 2, residues 275-279; region 5,residues 297-301; region 6, residues 329-334. Loops separate regions 1and 2, and regions 5 and 6.

The CUB domain of zveg3 is believed to form a beta barrel structure withnine distinct beta strand-like regions. These regions comprise residues48-51, 55-59, 72-78, 85-90, 92-94, 107-112, 119-123, 139-146, and156-163 of SEQ ID NO:2. A multiple alignment of CUB domains of Xenopuslaevis neuropilin precursor (Takagi et al., ibid.), human BMP-1 (Wozneyet al., ibid.), and X. laevis tolloid-like protein (Lin et al., ibid.)indicates the presence of a conserved motif corresponding to residues104-124 of SEQ ID NO:2. This motif is represented by the formulaC[KR]Y[DNE] [WYF]X{11,15}G[KR] [WYF]C (SEQ ID NO:4), wherein squarebrackets indicate the allowable residues at a given position and X{y,z}is as defined above.

The proteins of the present invention include proteins comprising CUBdomains homologous to the CUB domain of zvegf3. These homologous domainsare from 100 to 120 residues in length and comprise a motif of thesequence C[KR]Y[DNE] [WYF]X{11,15}G[KR] [WYF]C (SEQ ID NO:4)corresponding to residues 104-124 of SEQ ID NO:2. These homologous CUBdomains are at least 90% identical to residues 46-163 of SEQ ID NO:2 orat least 95% identical. to residues 46-163 of SEQ ID NO:2.

CUB domain-containing proteins of the present invention may furtherinclude a zvegf3 interdomain region or homolog thereof. The interdomainregion is at least 90% identical to residues 164 to 234 of SEQ ID NO:2.

Additional proteins of the present invention comprise the zvegf3 growthfactor domain or a homolog thereof. These proteins thus comprise apolypeptide segment that is at least 90% or 95% identical to residues235-345 of SEQ ID NO:2, wherein the polypeptide segment comprises Cysresidues at positions corresponding to residues 250, 280, 284, 296, 335,and 337 of SEQ ID NO:2; a glycine at a position corresponding to residue284 of SEQ ID NO:2; and the sequence motifCX{18,33}CXGXCX{6,33}CX{20,40}CXC (SEQ ID NO:3) corresponding toresidues 250-337 of SEQ ID NO:2.

Additional proteins comprising combinations of the CUB domain,interdomain region, and growth factor domain are shown above in Table 1.In each case, the invention also includes homologous proteins comprisinghomologous domains as disclosed above.

Structural analysis and homology predict that zvegf3 polypeptidescomplex with a second polypeptide to form multimeric proteins. Theseproteins include homodimers and heterodimers. In the latter case, thesecond polypeptide can be a truncated or other variant zvegf3polypeptide or another polypeptide, such as a PlGF, PDGF-A, PDGF-B,VEGF, VEGF-B, VEGF-C, VEGF-D, or zvegf4 polypeptide. Among the dimericproteins within the present invention are dimers formed by non-covalentassociation (e.g., hydrophobic interactions) with a second subunit,either a second zvegf3 polypeptide or other second subunit, or bycovalent association stabilized by intermolecular disulfide bondsbetween cysteine residues of the component monomers. Within SEQ ID NO:2,the Cys residues at positions 274, 286, 287, 291, 294, and 339 may formintramolecular or intermolecular disulfide bonds. It is likely thatresidues 274 and 287 form interchain disulfide bonds. In homodimers, thecomponent polypeptides (designated A and B) may be joined in anantiparallel arrangement with a pattern of interchain disulfide bondsA274—B287, A287—B274. The data further suggest that additionalintrachain disulfides are formed by the pairing of Cys286 with Cys291,and Cys294 with Cys339, although some or all of these four residues maybe involved in interchain pairing.

The present invention thus provides a variety of multimeric proteinscomprising a zvegf3 polypeptide as disclosed above. These zvegf3polypeptides include zvegf3₁₅₋₂₃₄, zvegf3₄₆₋₂₃₄, zvegf3₁₅₋₂₂₉ amide,zvegf3₁₅₋₂₃₀, zvegf3₁₅₋₃₄₅, zvegf3₄₆₋₃₄₅, and zvegf3₂₃₅₋₃₄₅, as well asvariants and derivatives of these polypeptides as disclosed herein.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603-616, 1986, andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-10919, 1992.Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 2 (amino acids are indicated by the standard one-lettercodes). The percent identity is then calculated as:$\frac{{Total}\quad {number}\quad {of}\quad {identical}\quad {matches}}{\begin{matrix}\left\lbrack {{length}\quad {of}\quad {the}\quad {longer}\quad {sequence}\quad {plus}\quad {the}} \right. \\{{number}\quad {of}\quad {gaps}\quad {introduced}\quad {into}\quad {the}\quad {longer}} \\\left. {{sequence}\quad {in}\quad {order}\quad {to}\quad {align}\quad {the}\quad {two}\quad {sequences}} \right\rbrack\end{matrix}} \times 100$

TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A  4 R −1  5 N −2  0  6D −2 −2  1  6 C  0 −3 −3 −3  9 Q −1  1  0  0 −3  5 E −1  0  0  2 −4  2 5 G  0 −2  0 −1 −3 −2 −2  6 H −2  0  1 −1 −3  0  0 −2  8 I −1 −3 −3 −3−1 −3 −3 −4 −3  4 L −1 −2 −3 −4 −1 −2 −3 −4 −3  2  4 K −1  2  0 −1 −3  1 1 −2 −1 −3 −2  5 M −1 −1 −2 −3 −1  0 −2 −3 −2  1  2 −1  5 F −2 −3 −3 −3−2 −3 −3 −3 −1  0  0 −3  0  6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2−4  7 S  1 −1  1  0 −1  0  0  0 −1 −2 −2  0 −1 −2 −1  4 T  0 −1  0 −1 −1−1 −1 −2 −2 −1 −1 −1 −1 −2 −1  1  5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2−3 −1  1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3  2 −1 −1 −2 −1  3 −3 −2−2  2  7 V  0 −3 −3 −3 −1 −2 −2 −3 −3  3  1 −2  1 −1 −2 −2  0 −3 −1 4

The level of identity between amino acid sequences can be determinedusing the “FASTA” similarity search algorithm disclosed by Pearson andLipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and by Pearson (Meth.Enzymol. 183:63, 1990). Briefly, FASTA first characterizes sequencesimilarity by identifying regions shared by the query sequence (e.g.,SEQ ID NO:2) and a test sequence that have either the highest density ofidentities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), whichallows for amino acid insertions and deletions. Preferred parameters forFASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, 1990 (ibid.).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

The present invention includes polypeptides having one or moreconservative amino acid changes as compared with the amino acid sequenceof SEQ ID NO:2. The BLOSUM62 matrix (Table 2) is an amino acidsubstitution matrix derived from about 2,000 local multiple alignmentsof protein sequence segments, representing highly conserved regions ofmore than 500 groups of related proteins (Henikoff and Henikoff, ibid.).Thus, the BLOSUM62 substitution frequencies can be used to defineconservative amino acid substitutions that may be introduced into theamino acid sequences of the present invention. As used herein, the term“conservative amino acid substitution” refers to a substitutionrepresented by a BLOSUM62 value of greater than −1. For example, anamino acid substitution is conservative if the substitution ischaracterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least one 1 (e.g., 1, 2 or 3), while more preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 2 (e.g., 2 or 3).

The proteins of the present invention can further comprise amino- orcarboxyl-terminal extensions, such as an amino-terminal methionineresidue, an amino or carboxyl-terminal cysteine residue to facilitatesubsequent linking to maleimide-activated keyhole limpet hemocyanin, asmall linker peptide of up to about 20-25 residues, or a polypeptideextension that facilitates purification (an affinity tag) as disclosedabove. Two or more affinity tags may be used in combination.Polypeptides comprising affinity tags can further comprise a polypeptidelinker and/or a proteolytic cleavage site between the zvegf3 polypeptideand the affinity tag. Exemplary cleavage sites include thrombin cleavagesites and factor Xa cleavage sites.

The present invention further provides a variety of other polypeptidefusions and related multimeric proteins comprising one or morepolypeptide fusions. For example, a zvegf3 polypeptide can be preparedas a fusion to a dimerizing protein as disclosed in U.S. Pat. Nos.5,155,027 and 5,567,584. Exemplary dimerizing proteins in this regardinclude immunoglobulin constant region domains. Dimerization can also bestabilized by fusing a zvegf3 polypeptide to a leucine zipper sequence(Riley et al., Protein Eng. 9:223-230, 1996; Mohamed et al., J. SteroidBiochem. Mol. Biol. 51:241-250, 1994). Immunoglobulin-zvegf3 polypeptidefusions and leucine zipper fusions can be expressed in geneticallyengineered cells to produce a variety of multimeric zvegf3 analogs.Auxiliary domains can be fused to zvegf3 polypeptides to target them tospecific cells, tissues, or macromolecules (e.g., collagen). Forexample, a zvegf3 polypeptide or protein can be targeted to apredetermined cell type by fusing a zvegf3 polypeptide to a ligand thatspecifically binds to a receptor on the surface of the target cell. Inthis way, polypeptides and proteins can be targeted for therapeutic ordiagnostic purposes. A zvegf3 polypeptide can be fused to two or moremoieties, such as an affinity tag for purification and a targetingdomain. Polypeptide fusions can also comprise one or more cleavagesites, particularly between domains. See, Tuan et al., Connective TissueResearch 34:1-9, 1996.

Polypeptide fusions of the present invention will generally contain notmore than about 1,500 amino acid residues, often not more than about1,200 residues, often not more than about 1,000 residues, and will inmany cases be considerably smaller. For example, a zvegf3 polypeptide of331 residues (residues 15-345 of SEQ ID NO:2) can be fused to E. coliβ-galactosidase (1,021 residues; see Casadaban et al., J. Bacteriol.143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xacleavage site to yield a polypeptide of 1366 residues. In a secondexample, residues 235-345 of SEQ ID NO:2 can be fused to maltose bindingprotein (approximately 370 residues), a 4-residue cleavage site, and a6-residue polyhistidine tag.

A polypeptide comprising the zvegf3 growth factor domain (e.g.,zvegf3₂₃₅₋₃₄₅ or zvegf3₁₆₄₋₃₄₅) can be fused to a non-zvegf3 CUB domain.Within a related embodiment of the invention, a zvegf3 polypeptidecomprising zvegf3 growth factor and CUB domains is fused to a non-zvegf3CUB domain, such as a CUB-domain comprising neuropilin polypeptide.

The present invention further provides polypeptide fusions comprisingthe zvegf3 CUB domain (e.g., zvegf3₄₅₋₁₆₃). The CUB domain, with itshomology to neuropilin-1, may be used to target zvegf3 or other proteinscontaining it to cells having cell-surface semaphorins, includingendothelial cells, neuronal cells, lymphocytes, and tumor cells. Thezvegf3 CUB domain can thus be joined to other moities, includingpolypeptides (e.g., other growth factors, antibodies, and enzymes) andnon-peptidic moieties (e.g., radionuclides, contrast agents, and thelike), to target them to cells expressing cell-surface semaphorins. Thedibasic and furin-like sites between the CUB and growth factor domainsof zvegf3 may allow for proteolytic release of the growth factor domainor other moiety through existing local proteases within tissues, or byproteases added from exogenous sources. The release of the targettedmoiety may provide more localized biological effects.

Proteins comprising the wild-type zvegf3 CUB domain and variants thereofmay be used to modulate activities mediated by cell-surface semaphorins.While not wishing to be bound by theory, zvegf3 may bind to semaphorinsvia its CUB domain. The observation that semaphorin III is involved invascular development suggests that members of the vascular growth factorfamily of proteins may also be involved, especially due to theco-binding activity of VEGF and semaphorin III to neuropilin-1. Zvegf3may thus be used to design agonists and antagonist ofneuropilin-semaphorin interactions. For example, the zvegf3 sequencedisclosed herein provides a starting point for the design of moleculesthat antagonize semaphorin-stimulated activities, including neuritegrowth, cardiovascular development, cartilage and limb development, andT and B-cell function. Additional applications include intervention invarious pathologies, including rheumatoid arthritis, various forms ofcancer, autoimmune disease, inflammation, retinopathies, hemangiomas,ischemic events within tissues including the heart, kidney andperipheral arteries, neuropathies, acute nerve damage, and diseases ofthe central and peripheral nervous systems.

Amino acid sequence changes are made in zvegf3 polypeptides so as tominimize disruption of higher order structure essential to biologicalactivity. In general, conservative amino acid changes are preferred.Changes in amino acid residues will be made so as not to disrupt thecystine knot and “bow tie” arrangement of loops in the growth factordomain that is characteristic of the protein family. Conserved motifswill also be maintained. The effects of amino acid sequence changes canbe predicted by computer modeling as disclosed above or determined byanalysis of crystal structure (see, e.g., Lapthorn et al., ibid.). Ahydrophobicity profile of SEQ ID NO:2 is shown in FIG. 1. Those skilledin the art will recognize that this hydrophobicity will be taken intoaccount when designing alterations in the amino acid sequence of azvegf3 polypeptide, so as not to disrupt the overall profile. Additionalguidance in selecting amino acid subsitutions is provided by thealignment of mouse and human zvegf3 sequences shown in FIG. 6.

The polypeptides of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, and 4-fluorophenylalanine. Several methods are knownin the art for incorporating non-naturally occurring amino acid residuesinto proteins. For example, an in vitro system can be employed whereinnonsense mutations are suppressed using chemically aminoacylatedsuppressor tRNAs. Methods for synthesizing amino acids andaminoacylating tRNA are known in the art. Transcription and translationof plasmids containing nonsense mutations is carried out in a cell-freesystem comprising an E. coli S30 extract and commercially availableenzymes and other reagents. Proteins are purified by chromatography.See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991;Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science259:806-809, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA90:10145-10149, 1993). In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem.271:19991-19998, 1996). Within a third method, E. coli cells arecultured in the absence of a natural amino acid that is to be replaced(e.g., phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. See, Koide et al., Biochem. 33:7470-7476, 1994.Naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions (Wynn and Richards, ProteinSci. 2:395-403, 1993).

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244, 1081-1085, 1989; Bass et al., Proc. Natl. Acad.Sci. USA 88:4498-4502, 1991). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity of otherproperties to identify amino acid residues that are critical to theactivity of the molecule.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53-57, 1988) or Bowie and Sauer(Proc. Natl. Acad. Sci. USA 86:2152-2156, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (e.g., Lowman et al., Biochem. 30:10832-10837, 1991;Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO92/06204) and region-directed mutagenesis (Derbyshire et al., Gene46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants of the disclosed zvegf3 DNA and polypeptide sequences can begenerated through DNA shuffling as disclosed by Stemmer, Nature370:389-391, 1994 and Stemmer, Proc. Natl. Acad. Sci. USA91:10747-10751, 1994. Briefly, variant genes are generated by in vitrohomologous recombination by random fragmentation of a parent genefollowed by reassembly using PCR, resulting in randomly introduced pointmutations. This technique can be modified by using a family of parentgenes, such as allelic variants or genes from different species, tointroduce additional variability into the process. Selection orscreening for the desired activity, followed by additional iterations ofmutagenesis and assay provides for rapid “evolution” of sequences byselecting for desirable mutations while simultaneously selecting againstdetrimental changes.

Mutagenesis methods as disclosed above can be combined with high volumeor high-throughput screening methods to detect biological activity ofzvegf3 variant polypeptides, in particular biological activity inmodulating cell proliferation or cell differentiation. For example,mitogenesis assays that measure dye incorporation or ³H-thymidineincorporation can be carried out on large numbers of samples, as cancell-based assays that detect expression of a reporter gene (e.g., aluciferase gene). Mutagenesis of the CUB domain can be used to modulateits binding to members of the semaphorin family, including enhancing orinhibiting binding to selected family members. A modified spectrum ofbinding activity may be desirable for optimizing therapeutic and/ordiagnostic utility of proteins comprising a zvegf3 CUB domain. Directbinding utilizing labeled CUB protein can be used to monitor changes inCUB domain binding activity to selected semaphorin family members.Semaphorins of interest in this regard include isolated proteins,proteins present in cell membranes, and proteins present oncell-surfaces. The CUB domain can be labeled by a variety of methodsincluding radiolabeling with isotopes, such as ¹²⁵I, conjugation toenzymes such as alkaline phosphatase or horseradish peroxidase,conjugation with biotin, and conjugation with various fluorescentmarkers including FITC. These and other assays are disclosed in moredetail below. Mutagenized DNA molecules that encode active zvegf3polypeptides can be recovered from the host cells and rapidly sequencedusing modern equipment. These methods allow the rapid determination ofthe importance of individual amino acid residues in a polypeptide ofinterest, and can be applied to polypeptides of unknown structure.

Using the methods discussed above, one of ordinary skill in the art canidentify and/or prepare a variety of polypeptides that are homologous tothe zvegf3 polypeptides disclosed above in Table 1 and retain thebiological properties of the wild-type protein. Such polypeptides canalso include additional polypeptide segments as generally disclosedabove.

The present invention also provides polynucleotide molecules, includingDNA and RNA molecules, that encode the zvegf3 polypeptides disclosedabove. The polynucleotides of the present invention include the sensestrand; the anti-sense strand; and the DNA as double-stranded, havingboth the sense and anti-sense strand annealed together by hydrogenbonds. A representative DNA sequences encoding zvegf3 polypeptides isset forth in SEQ ID NO:1. Additional DNA sequences encoding zvegf3polypeptides can be readily generated by those of ordinary skill in theart based on the genetic code. Counterpart RNA sequences can begenerated by substitution of U for T.

Those skilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among polynucleotide molecules encoding zvegf3 polypeptides.SEQ ID NO:6 is a degenerate DNA sequence that encompasses all DNAs thatencode the zvegf3 polypeptide of SEQ ID NO: 2. Those skilled in the artwill recognize that the degenerate sequence of SEQ ID NO:6 also providesall RNA sequences encoding SEQ ID NO:2 by substituting U for T. Thus,zvegf3 polypeptide-encoding polynucleotides comprising nucleotides1-1035, 1-489, 43-489, 136-489, 43-702, 136-702, 43-690, 43-1035,136-1035, and 703-1035 of SEQ ID NO:6 and their RNA equivalents arecontemplated by the present invention. Table 3 sets forth the one-lettercodes used within SEQ ID NO:6 to denote degenerate nucleotide positions.“Resolutions” are the nucleotides denoted by a code letter. “Complement”indicates the code for the complementary nucleotide(s). For example, thecode Y denotes either C or T, and its complement R denotes A or G, Abeing complementary to T, and G being complementary to C.

TABLE 3 Nucleotide Resolutions Complement Resolutions A A T T C C G G GG C C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G SC|G W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|TH A|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NO:6, encompassing all possiblecodons for a given amino acid, are set forth in Table 4, below.

TABLE 4 One- Amino Letter Degenerate Acid Code Condons Condon Cys C TGCTGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN ProP CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGTGGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAGCAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAGAAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTGYTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp WTGG TGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN Gap— — — —

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequences may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NO: 2. Variant sequences can be readily tested forfunctionality as described herein.

Within certain embodiments of the invention the isolated polynucleotideswill hybridize to similar sized regions of SEQ ID NO:1, or a sequencecomplementary thereto, under stringent conditions. In general, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence at a defined ionic strength andpH. The T_(m) is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Typical stringent conditions are those in which the saltconcentration is up to about 0.03 M at pH 7 and the temperature is atleast about 60° C.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. Complementary DNA (cDNA) clones are prepared fromRNA that is isolated from a tissue or cell that produces large amountsof zvegf3 RNA. Such tissues and cells are identified by Northernblotting (Thomas, Proc. Natl. Acad. Sci. USA 77:5201, 1980), and includethyroid, spinal cord, and adrenal gland. Total RNA can be prepared usingguanidine HCl extraction followed by isolation by centrifugation in aCsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)⁺RNA is prepared from total RNA using the method of Aviv and Leder (Proc.Natl. Acad. Sci. USA 69:1408-1412, 1972). Complementary DNA (cDNA) isprepared from poly(A)⁺ RNA using known methods. In the alternative,genomic DNA can be isolated. For some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods foridentifying and isolating cDNA and genomic clones are well known andwithin the level of ordinary skill in the art, and include the use ofthe sequence disclosed herein, or parts thereof, for probing or priminga library. Polynucleotides encoding zvegf3 polypeptides are identifiedand isolated by, for example, hybridization or polymerase chain reaction(“PCR”, Mullis, U.S. Pat. No. 4,683,202). Expression libraries can beprobed with antibodies to zvegf3, receptor fragments, or other specificbinding partners.

Those skilled in the art will recognize that the sequences disclosed inSEQ ID NOS: 1 and 2 represent a single allele of human zvegf3. Allelicvariants of these sequences can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Alternatively spliced forms of zvegf3 are also expected to exist.

The zvegf3 polynucleotide sequence disclosed herein can be used toisolate polynucleotides encoding other zvegf3 proteins. Such otherpolynucleotides include allelic variants, alternatively spliced cDNAsand counterpart polynucleotides from other species (orthologs). Theseorthologous polynucleotides can be used, inter alia, to prepare therespective orthologous proteins. Other species of interest include, butare not limited to, mammalian, avian, amphibian, reptile, fish, insectand other vertebrate and invertebrate species. Of particular interestare zvegf3 polynucleotides and proteins from other mammalian species,including non-human primate, murine, porcine, ovine, bovine, canine,feline, and equine polynucleotides and proteins. Non-human ztbg1polypeptides and polynucleotides, as well as antagonists thereof andother related molecules, can be used, inter alia, in veterinarymedicine. orthologs of human zvegf3 can be cloned using information andcompositions provided by the present invention in combination withconventional cloning techniques. For example, a cDNA can be cloned usingmRNA obtained from a tissue or cell type that expresses zvegf3 asdisclosed herein. Suitable sources of mRNA can be identified by probingNorthern blots with probes designed from the sequences disclosed herein.A library is then prepared from mRNA of a positive tissue or cell line.A zvegf3-encoding cDNA can then be isolated by a variety of methods,such as by probing with a complete or partial human cDNA or with one ormore sets of degenerate probes based on the disclosed sequences.Hybridization will generally be done under low stringency conditions,wherein washing is carried out in 1×SSC with an initial wash at 40° C.and with subsequent washes at 5° C. higher intervals until background issuitably reduced. A cDNA can also be cloned using the polymerase chainreaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primersdesigned from the representative human zvegf3 sequence disclosed herein.Within an additional method, the cDNA library can be used to transformor transfect host cells, and expression of the cDNA of interest can bedetected with an antibody to zvegf3 polypeptide. Similar techniques canalso be applied to the isolation of genomic clones.

For any zvegf3 polypeptide, including variants and fusion proteins, oneof ordinary skill in the art can readily generate a fully degeneratepolynucleotide sequence encoding that variant using the information setforth in Tables 3 and 4, above.

Conserved regions of zvegf3, identified by alignment with sequences ofother family members, can be used to identify related polynucleotidesand proteins. For instance, reverse transcription-polymerase chainreaction (RT-PCR) and other techniques known in the art can be used toamplify sequences encoding the conserved motifs present in zvegf3 fromRNA obtained from a variety of tissue sources. In particular, highlydegenerate primers as shown below in Table 5 (designed from an alignmentof zvegf3 with PDGF A and B chains, VEGF, VEGF-B, VEGF-C, and VEGF-D)are useful for cloning polynucleotides encoding homologous growth factordomains. Primers shown in Table 6, designed from an alignment of zvegf3with X. laevis neuropilin precursor, human BMP-1, and X. laevistolloid-like protein, are useful for cloning polynucleotides encodingCUB domains. The primers of Tables 6 and 7 can thus be used to obtainadditional polynucleotides encoding homologs of the zvegf3 sequence ofSEQ ID NO:1 and NO:2.

TABLE 5 zvegf3 residues 279-284 degenerate: MGN TGY GGN GGN AAY TG (SEQID NO:7) consensus: MGN TGY DSN GGN WRY TG (SEQ ID NO:8) complement: CARYWN CCN SHR CAN CK (SEQ ID NO:9) zvegf3 residues 270-275 degenerate: TTYTGG CCN GGN TGY YT (SEQ ID NO:10) consensus: NTN DDN CCN NSN TGY BT (SEQID NO:11) complement: AVR CAN SNN GGN HHN AN (SEQ ID NO:12) zvegf3residues 332-337 degenerate CAY GAR GAR TGY GAY TG (SEQ ID NO:13)consensus: CAY NNN NVN TGY VVN TG (SEQ ID NO:14) complement: CAN BBR CANBNN NNR TG (SEQ ID NO:15) zvegf3 residues 250-255 degenerate: TGY ACNCCN MGN AAY TT (SEQ ID NO:16) consensus: TGY HNN MCN MKN RMN DH (SEQ IDNO:17) complement: DHN KYN MKN GKN NDR CA (SEQ ID NO:18)

TABLE 6 zvegf3 residues 104-109 consensus: TGY AAR TAY GAY TWY GT (SEQID NO:19) complement: ACR WAR TCR TAY TTR CA (SEQ ID NO:20) zvegf3residues 120-125 consensus: YWN GGN MRN TDB TGY GG (SEQ ID NO:21)complement: CCR CAV HAN YKN CCN WR (SEQ ID NO:22) zvegf3 residues 63-68consensus: TDB CCN MAN DVN TAY CC (SEQ ID NO:23) complement: GGR TAN BHNTKN GGV HA (SEQ ID NO:24)

Zvegf3 polynucleotide sequences disclosed herein can also be used asprobes or primers to clone 5′ non-coding regions of a zvegf3 gene,including promoter sequences. These flanking sequences can be used todirect the expression of zvegf3 and other recombinant proteins. Inaddition, 5′ flanking sequences can be used as targetting sites forregulatory constructs to activate or increase expression of endogenouszvegf3 genes as disclosed by Treco et al., U.S. Pat. No. 5,641,670.

The polynucleotides of the present invention can also be prepared byautomated synthesis. The current method of choice is the phosphoramiditemethod. If chemically synthesized, double-stranded DNA is required, eachcomplementary strand is made separately. The production of short,double-stranded segments (60 to 80 bp) is technically straightforwardand can be accomplished by synthesizing the complementary strands andthen annealing them. Longer segments (typically >300 bp) are assembledin modular form from single-stranded fragments that are from 20 to 100nucleotides in length. Automated synthesis of polynucleotides is withinthe level of ordinary skill in the art, and suitable equipment andreagents are available from commercial suppliers. See, in general, Glickand Pasternak, Molecular Biotechnology, Principles & Applications ofRecombinant DNA, ASM Press, Washington, D.C., 1994; Itakura et al., Ann.Rev. Biochem. 53: 323-56, 1984; and Climie et al., Proc. Natl. Acad.Sci. USA 87:633-7, 1990.

The polypeptides of the present invention, including full-lengthpolypeptides, biologically active fragments, and fusion polypeptides canbe produced in genetically engineered host cells according toconventional techniques. Suitable host cells are those cell types thatcan be transformed or transfected with exogenous DNA and grown inculture, and include bacteria, fungal cells, and cultured highereukaryotic cells (including cultured cells of multicellular organisms).Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989, and Ausubel et al.,eds., Current Protocols in Molecular Biology, Green and Wiley and Sons,NY, 1993.

In general, a DNA sequence encoding a zvegf3 polypeptide is operablylinked to other genetic elements required for its expression, generallyincluding a transcription promoter and terminator; within an expressionvector. The vector will also commonly contain one or more selectablemarkers and one or more origins of replication, although those skilledin the art will recognize that within certain systems selectable markersmay be provided on separate vectors, and replication of the exogenousDNA may be provided by integration into the host cell genome. Selectionof promoters, terminators, selectable markers, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art. Many such elements are described in the literature andare available through commercial suppliers.

To direct a zvegf3 polypeptide into the secretory pathway of a hostcell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be that of zvegf3, or may be derivedfrom another secreted protein (e.g., t-PA; see, U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto the zvegf3 DNA sequence, i.e., the two sequences are joined in thecorrect reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe polypeptide of interest, although certain signal sequences may bepositioned elsewhere in the DNA sequence of interest (see, e.g., Welchet al., U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.5,143,830).

Expression of zvegf3 polypeptides via a host cell secretory pathway isexpected to result in the production of multimeric proteins. As notedabove, such multimers include both homomultimers and heteromultimers,the latter including proteins comprising only zvegf3 polypeptides andproteins including zvegf3 and heterologous polypeptides. For example, aheteromultimer comprising a zvegf3 polypeptide and a polypeptide from arelated family member (e.g., VEGF, VEGF-B, VEGF-C, VEGF-D, zvegf4, PlGF,PDGF-A, or PDGF-B) can be produced by co-expression of the twopolypeptides in a host cell. Sequences encoding these other familymembers are known. See, for example, Dvorak et al, ibid.; Olofsson etal, ibid.; Hayward et al., ibid.; Joukov et al., ibid.; Oliviero et al.,ibid.; Achen et al., ibid.; Maglione et al., ibid.; Heldin et al., U.S.Pat. No. 5,219,759; and Johnsson et al., ibid. If a mixture of proteinsresults from expression, individual species are isolated by conventionalmethods. Monomers, dimers, and higher order multimers are separated by,for example, size exclusion chromatography. Heteromultimers can beseparated from homomultimers by immunoaffinity chromatography usingantibodies specific for individual dimers or by sequentialimmunoaffinity steps using antibodies specific for individual componentpolypeptides. See, in general, U.S. Pat. No. 5,094,941. Multimers mayalso be assembled in vitro upon incubation of component polypeptidesunder suitable conditions. In general, in vitro assembly will includeincubating the protein mixture under denaturing and reducing conditionsfollowed by, refolding and reoxidation of the polypeptides to fromhomodimers and heterodimers. Recovery and assembly of proteins expressedin bacterial cells is disclosed below.

Cultured mammalian cells are suitable hosts for use within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-845, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7, 1987; Mackey etal., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988; Hawley-Nelson et al.,Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 199,3). The productionof recombinant polypeptides in cultured mammalian cells is disclosed by,for example, Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al.,U.S. Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Rockville, Md. Sstrongtranscription promoters include promoters from SV-40 or cytomegalovirus.See, e.g., U.S. Pat. No. 4,956,288. Other suitable promoters includethose from metallothionein genes (U.S. Pat. Nos. 4,579,821 and4,601,978) and the adenovirus major late promoter. Expression vectorsfor use in mammalian cells include pZP-1 and pZP-9, which have beendeposited with the American Type Culture Collection, Rockville, Md. USAunder accession numbers 98669 and 98668, respectively.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Anexemplary selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.An exemplary amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasecan be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, includinginsect cells, plant cells and avian cells. The use of Agrobacteriumrhizogenes as a vector for expressing genes in plant cells has beenreviewed by Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.Transformation of insect cells and production of foreign polypeptidestherein is disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPOpublication WO 94/06463.

Insect cells can be infected with recombinant baculovirus, commonlyderived from Autographa californica nuclear polyhedrosis virus (AcNPV).See, King and Possee, The Baculovirus Expression System: A LaboratoryGuide, London, Chapman & Hall; O'Reilly et al., Baculovirus ExpressionVectors: A Laboratory Manual, New York, Oxford University Press., 1994;and Richardson, Ed., Baculovirus Expression Protocols. Methods inMolecular Biology, Humana Press, Totowa, N.J., 1995. Recombinantbaculovirus can also be produced through the use of a transposon-basedsystem described by Luckow et al. (J. Virol. 67:4566-4579, 1993). Thissystem, which utilizes transfer vectors, is commercially available inkit form (Bac-to-Bac™ kit; Life Technologies, Rockville, Md.). Thetransfer vector (e.g., pFastBacl™; Life Technologies) contains a Tn7transposon to move the DNA encoding the protein of interest into abaculovirus genome maintained in E. coli as a large plasmid called a“bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 71:971-976, 1990;Bonning et al., J. Gen. Virol. 75:1551-1556, 1994; and Chazenbalk andRapoport, J. Biol. Chem. 270:1543-1549, 1995. In addition, transfervectors can include an in-frame fusion with DNA encoding a polypeptideextension or affinity tag as disclosed above. Using techniques known inthe art, a transfer vector containing a zvegf3-encoding sequence istransformed into E. coli host cells, and the cells are screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, such as Sf9 cells. Recombinant virus thatexpresses zvegf3 protein is subsequently produced. Recombinant viralstocks are made by methods commonly used the art.

For protein production, the recombinant virus is used to infect hostcells, typically a cell line derived from the fall armyworm, Spodopterafrugiperda (e.g., Sf9 or Sf21 cells) or Trichoplusia ni (e.g., HighFive™ cells; Invitrogen, Carlsbad, Calif.). See, in general, Glick andPasternak, Molecular Biotechnology: Principles and Applications ofRecombinant DNA, ASM Press, Washington, D.C., 1994. See also, U.S. Pat.No. 5,300,435. Serum-free media are used to grow and maintain the cells.Suitable media formulations are known in the art and can be obtainedfrom commercial suppliers. The cells are grown up from an inoculationdensity of approximately 2-5×10⁵ cells to a density of 1-2×10⁶ cells, atwhich time a recombinant viral stock is added at a multiplicity ofinfection (MOI) of 0.1 to 10, more typically near 3. Procedures used aregenerally described in available laboratory manuals (e.g., King andPossee, ibid.; O'Reilly et al., ibid.; Richardson, ibid.).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). An exemplary vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-3465, 1986; Cregg, U.S. Pat. No. 4,882,279; andRaymond et al., Yeast 14, 11-23, 1998. Aspergillus cells may be utilizedaccording to the methods of McKnight et al., U.S. Pat. No. 4,935,349.Methods for transforming Acremonium chrysogenum are disclosed by Suminoet al., U.S. Pat. No. 5,162,228. Methods for transforming Neurospora aredisclosed by Lambowitz, U.S. Pat. No. 4,486,533. Production ofrecombinant proteins in Pichia methanolica is disclosed in U.S. Pat.Nos. 5,716,808, 5,736,383, 5,854,039, and 5,888,768.

Prokaryotic host cells, including strains of the bacteria Escherichiacoli, Bacillus and other genera are also useful host cells within thepresent invention. Techniques for transforming these hosts andexpressing foreign DNA sequences cloned therein are well known in theart (see, e.g., Sambrook et al., ibid.). When expressing a zvegf3polypeptide in bacteria such as E. coli, the polypeptide may be retainedin the cytoplasm, typically as insoluble granules, or may be directed tothe periplasmic space by a bacterial secretion sequence. In the formercase, the cells are lysed, and the granules are recovered and denaturedusing, for example, guanidine isothiocyanate or urea. The denaturedpolypeptide can then be refolded and dimerized by diluting thedenaturant, such as by dialysis against a solution of urea and acombination of reduced and oxidized glutathione, followed by dialysisagainst a buffered saline solution. In the alternative, the protein maybe recovered from the cytoplasm in soluble form and isolated without theuse of denaturants. The protein is recovered from the cell as an aqueousextract in, for example, phosphate buffered saline. To capture theprotein of interest, the extract is applied directly to achromatographic medium, such as an immobilized antibody orheparin-Sepharose column. Secreted polypeptides can be recovered fromthe periplasmic space in a soluble and functional form by disrupting thecells (by, for example, sonication or osmotic shock) to release thecontents of the periplasmic space and recovering the protein, therebyobviating the need for denaturation and refolding.

Production of zvegf3 fusion proteins in prokaryotic host cells is ofparticular interest. An exemplary such fusion protein comprises a zvegf3polypeptide fused to maltose binding protein. Such fusions may furthercomprise additional sequences, such as polyhistidine to provide foraffinity purification of the polypeptide fusion. An enzymatic cleavagesite (e.g., a thrombin cleavage site) may also be included to allow forseparation of zvegf3 and non-zvegf3 components of the fusion.

Transformed or transfected host cells are cultured according toconventional procedures in a culture medium containing nutrients andother components required for the growth of the chosen host cells. Avariety of suitable media, including defined media and complex media,are known in the art and generally include a carbon source, a nitrogensource, essential amino acids, vitamins and minerals. Media may alsocontain such components as growth factors or serum, as required. Thegrowth medium will generally select for cells containing the exogenouslyadded DNA by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker carried on theexpression vector or co-transfected into the host cell. P. methanolicacells, for example, are cultured in a medium comprising adequate sourcesof carbon, nitrogen and trace nutrients at a temperature of about 25° C.to 35° C. Liquid cultures are provided with sufficient aeration byconventional means, such as shaking of small flasks or sparging offermentors.

Zvegf3 polypeptides or fragments thereof can also be prepared throughchemical synthesis according to methods known in the art, includingexclusive solid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989.

Non-covalent complexes comprising a zvegf3 polypeptide can be preparedby incubating a zvegf3 polypeptide and a second polypeptide (e.g., azvegf3 polypeptide or another peptide of the PDGF/VEGF family) atnear-physiological pH. In a typical reaction, polypeptides at aconcentration of about 0.1-0.5 μg/μl are incubated at pH≈7.4 in a weakbuffer (e.g., 0.01 M phosphate or acetate buffer); sodium chloride maybe included at a concentration of about 0.1 M. At 37° C. the reaction isessentially complete with 4-24 hours. See, for example, Weintraub etal., Endocrinology 101:225-235, 1997.

Covalent complexes can also be made by isolating the desired componentpolypeptides and combining them in vitro. Covalent complexes that can beprepared in this manner include homodimers of zvegf3 polypeptides,heterodimers of two different zvegf3 polypeptides, and heterodimers of azvegf3 polypeptide and a polypeptide from another family member of theVEGF/PDGF family of proteins. The two polypeptides are mixed togetherunder denaturing and reducing conditions, followed by renaturation ofthe proteins by removal of the denaturants. Removal can be done by, forexample, dialysis or size exclusion chromatography to provide for bufferexchange. When combining two different polypeptides, the resultingrenaturated proteins may form homodimers of the individual components aswell as heterodimers of the two polypeptide components. See, Cao et al.,J. Biol. Chem. 271:3154-3162, 1996.

Depending upon the intended use, polypeptides and proteins of thepresent invention can be purified to ≧80% purity, to ≧90% purity, to≧95% purity, or to a pharmaceutically pure state, that is greater than99.9% pure with respect to contaminating macromolecules, particularlyother proteins and nucleic acids, and free of infectious and pyrogenicagents.

Expressed recombinant zvegf3 proteins (including chimeric polypeptidesand multimeric proteins) are purified by conventional proteinpurification methods, typically by a combination of chromatographictechniques. See, in general, Affinity Chromatography: Principles &Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988; and Scopes,Protein Purification: Principles and Practice, Springer-Verlag, NewYork, 1994. Proteins comprising a polyhistidine affinity tag (typicallyabout 6 histidine residues) are purified by affinity chromatography on anickel chelate resin. See, for example, Houchuli et al., Bio/Technol. 6:1321-1325, 1988. Furthermore, the growth factor domain itself binds tonickel resin at pH 7.0-8.0 and 25 mM Na phosphate, 0.25 M NaCl. Boundprotein can be eluted with a descending pH gradient down to pH 5.0 or animidazole gradient. Proteins comprising a glu-glu tag can be purified byimmunoaffinity chromatography according to conventional procedures. See,for example, Grussenmeyer et al., ibid. Maltose binding protein fusionsare purified on an amylose column according to methods known in the art.As disclosed in more detail below, zvegf3 growth factor domain proteincan be purified using a combination of chromatography on a strong cationexchanger followed by hydrophobic interaction chromatography. When theprotein is produced in BHK cells, insulin-like growth factor bindingprotein 4 (IGFBP4) co-purifies with the zvegf3 under these conditions.Further purification can be obtained using reverse-phase HPLC, anionexchange on a quaternary amine strong cation exchanger at low ionicstrength and pH from 7.0 to 9.0, or hydrophobic interactionchromatography on a phenyl ether resin. It has also been found thatzvegf3 binds to various dye matrices (e.g., BLUE1, BLUE 2, ORANGE 1,ORANGE 3, and RED3 from Lexton Scientific, Signal Hill, Calif.) in PBSat pH 6-8, from which the bound protein can be eluted in 1-2M NaCl in 20mM boric acid buffer at pH 8.8. Protein eluted from RED3 may be passedover RED2 (Lexton Scientific) to remove remaining contaminants.

Using methods known in the art, zvegf3 proteins can be prepared asmonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

The invention further provides polypeptides that comprise anepitope-bearing portion of a protein as shown in SEQ ID NO:2. An“epitope” is a region of a protein to which an antibody can bind. See,for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002,1984. Epitopes can be linear or conformational, the latter beingcomposed of discontinuous regions of the protein that form an epitopeupon folding of the protein. Linear epitopes are generally at least 6amino acid residues in length. Relatively short synthetic peptides thatmimic part of a protein sequence are routinely capable of eliciting anantiserum that reacts with the partially mimicked protein. See,Sutcliffe et al., Science 219:660-666, 1983. Antibodies that recognizeshort, linear epitopes are particularly useful in analytic anddiagnostic applications that employ denatured protein, such as Westernblotting (Tobin, Proc. Natl. Acad. Sci. USA 76:4350-4356, 1979).Antibodies to short peptides may also recognize proteins in nativeconformation and will thus be useful for monitoring protein expressionand protein isolation, and in detecting zvegf3 proteins in solution,such as by ELISA or in immunoprecipitation studies.

Antigenic, epitope-bearing polypeptides of the present invention areuseful for raising antibodies, including monoclonal antibodies, thatspecifically bind to a zvegf3 protein. Antigenic, epitope-bearingpolypeptides contain a sequence of at least six, often at least nine,more often from 15 to about 30 contiguous amino acid residues of azvegf3 protein (e.g., SEQ ID NO:2). Polypeptides comprising a largerportion of a zvegf3 protein, i.e. from 30 to 50 residues up to theentire sequence are included. It is preferred that the amino acidsequence of the epitope-bearing polypeptide is selected to providesubstantial solubility in aqueous solvents, that is the sequenceincludes relatively hydrophilic residues, and hydrophobic residues aresubstantially avoided. Such regions include residues 43-48, 96-101,97-102, 260-265, and 330-335 of SEQ ID NO:2. As noted above, it isgenerally preferred to use somewhat longer peptides as immunogens, suchas a peptide comprising residues 80-104, 299-314, and 299-326 of SEQ IDNO:2. The latter peptide can be prepared with an additional N-terminalcys residue to facilitate coupling.

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingnon-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Monoclonal antibodies can also beproduced in mice that have been genetically altered to produceantibodies that have a human structure.

Methods for preparing and isolating polyclonal and monoclonal antibodiesare well known in the art. See, for example, Cooligan, et al. (eds.),Current Protocols in Immunology, National Institutes of Health, JohnWiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor, N.Y., 1989; andHurrell, J. G. R. (ed.), Monoclonal Hybridoma Antibodies: Techniques andApplications, CRC Press, Inc., Boca Raton, Fla., 1982. As would beevident to one of ordinary skill in the art, polyclonal antibodies canbe generated from inoculating a variety of warm-blooded animals such ashorses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats witha zvegf3 polypeptide or a fragment thereof. The immunogenicity of azvegf3 polypeptide may be increased through the use of an adjuvant, suchas alum (aluminum hydroxide) or Freund's complete or incompleteadjuvant. Polypeptides useful for immunization also include fusionpolypeptides, such as fusions of zvegf3 or a portion thereof with animmunoglobulin polypeptide or with maltose binding protein. If thepolypeptide portion is “hapten-like”, such portion may be advantageouslyjoined or linked to a macromolecular carrier (such as keyhole limpethemocyanin (KLH), bovine serum albumin (BSA), or tetanus toxoid) forimmunization.

Alternative techniques for generating or selecting antibodies include invitro exposure of lymphocytes to zvegf3 protein or peptide, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled zvegf3 protein orpeptide). Techniques for creating and screening such random peptidedisplay libraries are known in the art (Ladner et al., U.S. Pat. No.5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner et al., U.S.Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No. 5,571,698), andrandom peptide display libraries and kits for screening such librariesare available commercially, for instance from Clontech Laboratories(Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New EnglandBiolabs, Inc. (Beverly, Mass.), and Pharmacia LKB Biotechnology Inc.(Piscataway, N.J.). Random peptide display libraries can be screenedusing the zvegf3 sequences disclosed herein to identify proteins whichbind to zvegf3. These “binding proteins”, which interact with zvegf3polypeptides, can be used for tagging cells or for isolating homologouspolypeptides by affinity purification, or they can be directly orindirectly conjugated to drugs, toxins, radionuclides, and the like.These binding proteins can also be used in analytical methods, such asfor screening expression libraries and neutralizing activity; withindiagnostic assays, such as for determining circulating levels ofpolypeptides; for detecting or quantitating soluble polypeptides asmarkers of underlying pathology or disease; and as zvegf3 antagonists toblock zvegf3 binding and signal transduction in vitro and in vivo.

Antibodies are determined to be specifically binding if they bind to azvegf3 polypeptide, peptide or epitope with an affinity at least 10-foldgreater than the binding affinity to control (non-zvegf3) polypeptide orprotein. In this regard, a “non-zvegf3 polypeptide” includes the relatedmolecules VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF-A, and PDGF-B, butexcludes zvegf3 polypeptides from non-human species. Due to the highlevel of amino acid sequence identity expected between zvegf3 orthologs,antibodies specific for human zvegf3 may also bind to zvegf3 from otherspecies. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949). Methods forscreening and isolating specific antibodies are well known in the art.See, for example, Paul (ed.), Fundamental Immunology, Raven Press, 1993;Getzoff et al., Adv. in Immunol. 43:1-98, 1988; Goding, J. W. (ed.),Monoclonal Antibodies: Principles and Practice, Academic Press Ltd.,1996; Benjamin et al., Ann. Rev. Immunol. 2:67-101, 1984.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to zvegf3 proteins orpeptides. Exemplary assays are described in detail in Antibodies: ALaboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor LaboratoryPress, 1988. Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation,enzyme-linked immunosorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. In addition,antibodies can be screened for binding to wild-type versus mutant zvegf3protein or polypeptide.

Antibodies to zvegf3 may be used for tagging cells that express zvegf3;for isolating zvegf3 by affinity purification; for diagnostic assays fordetermining circulating levels of zvegf3 polypeptides; for detecting orquantitating soluble zvegf3 as a marker of underlying pathology ordisease; in analytical methods employing FACS; for screening expressionlibraries; for generating anti-idiotypic antibodies; and as neutralizingantibodies or as antagonists to block zvegf3 activity in vitro and invivo. Suitable direct tags or labels include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles and the like; indirect tags or labels mayfeature use of biotin-avidin or other complement/anti-complement pairsas intermediates. Antibodies may also be directly or indirectlyconjugated to drugs, toxins, radionuclides and the like, and theseconjugates used for in vivo diagnostic or therapeutic applications.Moreover, antibodies to zvegf3 or fragments thereof may be used in vitroto detect denatured zvegf3 or fragments thereof in assays, for example,Western Blots or other assays known in the art. Antibodies can also beused to target an attached therapeutic or diagnostic moiety to cellsexpressing zvegf3 or receptors for zvegf3.

For some applications (e.g., certain therapeutic applications) it ispreferred to use neutralizing antibodies. As used herein, the term“neutralizing antibody” denotes an antibody that inhibits at least 50%of the biological activity of the cognate antigen when the antibody isadded at a 1000-fold molar access. Those of skill in the art willrecognize that greater neutralizing activity is sometimes desirable, andantibodies that provide 50% inhibition at a 100-fold or 10-fold molaraccess may be advantageously employed.

Activity of zvegf3 proteins and antagonists thereof can be measured invitro using cultured cells or in vivo by administering molecules of theclaimed invention to an appropriate animal model. Target cells for usein zvegf3 activity assays include vascular cells (especially endothelialcells and smooth muscle cells), hematopoietic (myeloid and lymphoid)cells, liver cells (including hepatocytes, fenestrated endothelialcells, Kupffer cells, and Ito cells), fibroblasts (including humandermal fibroblasts and lung fibroblasts), neurite cells (includingastrocytes, glial cells, dendritic cells, and PC-12 cells), Schwanncells, fetal lung cells, articular synoviocytes, pericytes,chondrocytes, oligodendrocytes, osteoblasts, and other cells expressingPDGF alpha receptors.

Zvegf3 proteins can be analyzed for receptor binding activity by avariety of methods well known in the art, including receptor competitionassays (Bowen-Pope and Ross, Methods Enzymol. 109:69-100, 1985), use ofsoluble receptors, and use of receptors produced as IgG fusion proteins(U.S. Pat. No. 5,750,375). Receptor binding assays can be performed oncell lines that contain known cell-surface receptors for evaluation. Thereceptors can be naturally present in the cell, or can be recombinantreceptors expressed by genetically engineered cells. Cell types that areable to bind zvegf3 can be identified through the use of zvegf3-toxinconjugates, such as conjugates of a zvegf3 protein and saporin. Bindingof the zvegf3-toxin conjugate by cells, either in tissue culture, inorgan cultures, or in vivo settings will allow for the incorporation ofthe conjugate into the cell. Once inside the cell saporin has a toxiceffect on the cell, thereby killing it. This activity can be used toidentify cell types that are able to bind and internalize zvegf3. Inaddition to allowing for the identification of responsive cell types,toxin conjugates can be used in in vivo studies to identify organs andtissues where zvegf3 has a biological activity by looking for pathologywithin the animal following injection of the conjugate.

Activity of zvegf3 proteins can be measured in vitro using culturedcells. Mitogenic activity can be measured using known assays, including³H-thymidine incorporation assays (as disclosed by, e.g., Raines andRoss, Methods Enzymol. 109:749-773, 1985 and Wahl et al., Mol. CellBiol. 8:5016-5025, 1988), dye incorporation assays (as disclosed by, forexample, Mosman, J. Immunol. Meth. 65:55-63, 1983 and Raz et al., ActaTrop. 68:139-147, 1997) or cell counts. Suitable mitogenesis assaysmeasure incorporation of ³H-thymidine into (1) 20% confluent cultures tolook for the ability of zvegf3 proteins to further stimulateproliferating cells, and (2) quiescent cells held at confluence for 48hours to look for the ability of zvegf3 proteins to overcomecontact-induced growth inhibition. Suitable dye incorporation assaysinclude measurement of the incorporation of the dye Alamar blue (Raz etal., ibid.) into target cells. See also, Gospodarowicz et al., J. Cell.Biol. 70:395-405, 1976; Ewton and Florini, Endocrinol. 106:577-583,1980; and Gospodarowicz et al., Proc. Natl. Acad. Sci. USA 86:7311-7315,1989. Cell differentiation can be assayed using suitable precursor cellsthat can be induced to differentiate into a more mature phenotype. Forexample, endothelial cells and hematopoietic cells are derived from acommon ancestral cell, the hemangioblast (Choi et al., Development125:725-732, 1998). Mesenchymal stem cells can also be used to measurethe ability of zvegf3 protein to stimulate differentiation intoosteoblasts. Differentiation is indicated by the expression ofosteocalcin, the ability of the cells to mineralize, and the expressionof alkaline phosphatase, all of which can be measured by routine methodsknown in the art. Effects of zvegf3 proteins on tumor cell growth andmetastasis can be analyzed using the Lewis lung carcinoma model, forexample as described by Cao et al. J. Exp. Med. 182:2069-2077, 1995.Activity of zvegf3 proteins on cells of neural origin can be analyzedusing assays that measure effects on neurite growth.

Zvegf 3 activity may also be detected using assays designed to measurezvegf3-induced production of one or more additional growth factors orother macromolecules. Such assays include those for determining thepresence of hepatocyte growth factor (HGF), epidermal growth factor(EGF), transforming growth factor alpha (TGFα), interleukin-6 (IL-6),VEGF, acidic fibroblast growth factor (aFGF), and angiogenin. Suitableassays include mitogenesis assays using target cells responsive to themacromolecule of interest, receptor-binding assays, competition bindingassays, immunological assays (e.g., ELISA), and other formats known inthe art. Metalloprotease secretion is measured from treated primaryhuman dermal fibroblasts, synoviocytes and chondrocytes. The relativelevels of collagenase, gelatinase and stromalysin produced in responseto culturing in the presence of a zvegf3 protein is measured usingzymogram gels (Loita and Stetler-Stevenson, Cancer Biology 1:96-106,1990). Procollagen/collagen synthesis by dermal fibroblasts andchondrocytes in response to a test protein is measured using ³H-prolineincorporation into nascent secreted collagen. ³H-labeled collagen isvisualized by SDS-PAGE followed by autoradiography (Unemori and Amento,J. Biol. Chem. 265: 10681-10685, 1990). Glycosaminoglycan (GAG)secretion from dermal fibroblasts and chondrocytes is measured using a1,9-dimethylmethylene blue dye binding assay (Farndale et al., Biochim.Biophys. Acta 883:173-177, 1986). Collagen and GAG assays are alsocarried out in the presence of IL-1β or TGF-β to examine the ability ofzvegf3 protein to modify the established responses to these cytokines.

Monocyte activation assays are carried out (1) to look for the abilityof zvegf3 proteins to further stimulate monocyte activation, and (2) toexamine the ability of zvegf3 proteins to modulate attachment-induced orendotoxin-induced monocyte activation (Fuhlbrigge et al., J. Immunol.138: 3799-3802, 1987). IL-1β and TNFα levels produced in response toactivation are measured by ELISA (Biosource, Inc. Camarillo, Calif.).Monocyte/macrophage cells, by virtue of CD14 (LPS receptor), areexquisitely sensitive to endotoxin, and proteins with moderate levels ofendotoxin-like activity will activate these cells.

Hematopoietic activity of zvegf3 proteins can be assayed on varioushematopoietic cells in culture. Suitable assays include primary bonemarrow or peripheral blood leukocyte colony assays, and later stagelineage-restricted colony assays, which are known in the art (e.g.,Holly et al., WIPO Publication WO 95/21920). Marrow cells plated on asuitable semi-solid medium (e.g., 50% methylcellulose containing 15%fetal bovine serum, 10% bovine serum albumin, and 0.6% PSN antibioticmix) are incubated in the presence of test polypeptide, then examinedmicroscopically for colony formation. Known hematopoietic factors areused as controls. Mitogenic activity of zvegf3 polypeptides onhematopoietic cell lines can be measured using ³H-thymidineincorporation assays, dye incorporation assays, or cell counts (Rainesand Ross, Methods Enzymol. 109:749-773, 1985 and Foster et al., U.S.Pat. No. 5,641,655). For example, cells are cultured in multi-wellmicrotiter plates. Test samples and ³H-thymidine are added, and thecells are incubated overnight at 37° C. Contents of the wells aretransferred to filters, dried, and counted to determine incorporation oflabel. Cell proliferation can also be measured using a calorimetricassay based on the metabolic breakdown of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)(Mosman, ibid.). Briefly, a solution of MTT is added to 100 μl of assaycells, and the cells are incubated at 37° C. After 4 hours, 200 μl of0.04 N HCl in isopropanol is added, the solution is mixed, and theabsorbance of the sample is measured at 570 nm.

Cell migration is assayed essentially as disclosed by Kähler et al.(Arteriosclerosis, Thrombosis, and Vascular Biology 17:932-939, 1997). Aprotein is considered to be chemotactic if it induces migration of cellsfrom an area of low protein concentration to an area of high proteinconcentration. The assay is performed using modified Boyden chamberswith a polystryrene membrane separating the two chambers (Transwell;Corning Costar Corp.). The test sample, diluted in medium containing 1%BSA, is added to the lower chamber of a 24-well plate containingTranswells. Cells are then placed on the Transwell insert that has beenpretreated with 0.2% gelatin. Cell migration is measured after 4 hoursof incubation at 37° C. Non-migrating cells are wiped off the top of theTranswell membrane, and cells attached to the lower face of the membraneare fixed and stained with 0.1% crystal violet. Stained cells are thenextracted with 10% acetic acid and absorbance is measured at 600 nm.Migration is then calculated from a standard calibration curve.

Smooth muscle cell (SMC) migration can be measured in the aortic explantassay of Kenagy et al. (Circulation 96:3555-3560, 1997). In a typicalprotocol, explants are prepared from baboon thoracic aortas, and theinner media is isolated and chopped into 1-mm² pieces. The explants areplaced in tissue culture flasks containing DMEM supplemented with 5μg/ml transferrin, 6 μg/ml insulin, 1 mg/ml ovalbumin, and the testcompound. The number of migrating cells is determined daily.

Cell adhesion activity is assayed essentially as disclosed by LaFleur etal. (J. Biol. Chem. 272:32798-32803, 1997). Briefly, microtiter platesare coated with the test protein, non-specific sites are blocked withBSA, and cells (such as smooth muscle cells, leukocytes, or endothelialcells) are plated at a density of approximately 10⁴-10⁵ cells/well. Thewells are incubated at 37° C. (typically for about 60 minutes), thennon-adherent cells are removed by gentle washing. Adhered cells arequantitated by conventional methods (e.g., by staining with crystalviolet, lysing the cells, and determining the optical density of thelysate). Control wells are coated with a known adhesive protein, such asfibronectin or vitronectin.

Assays for angiogenic activity are also known in the art. For example,the effect of zvegf3 proteins on primordial endothelial cells inangiogenesis can be assayed in the chick chorioallantoic membraneangiogenesis assay (Leung, Science 246:1306-1309, 1989; Ferrara, Ann. NYAcad. Sci. 752:246-256, 1995). Briefly, a small window is cut into theshell of an eight-day old fertilized egg, and a test substance isapplied to the chorioallantoic membrane. After 72 hours, the membrane isexamined for neovascularization. Other suitable assays includemicroinjection of early stage quail (Coturnix coturnix japonica) embryosas disclosed by Drake et al. (Proc. Natl. Acad. Sci. USA 92:7657-7661,1995); the rodent model of corneal neovascularization disclosed byMuthukkaruppan and Auerbach (Science 205:1416-1418, 1979), wherein atest substance is inserted into a pocket in the cornea of an inbredmouse; and the hampster cheek pouch assay (Höckel et al., Arch. Surg.128:423-429, 1993). Induction of vascular permeability, which isindicative of angiogenic activity, is measured in assays designed todetect leakage of protein from the vasculature of a test animal (e.g.,mouse or guinea pig) after administration of a test compound (Miles andMiles, J. Physiol. 118:228-257, 1952; Feng et al., J. Exp. Med.183:1981-1986, 1996). In vitro assays for angiogenic activity includethe tridimensional collagen gel matrix model (Pepper et al. Biochem.Biophys. Res. Comm. 189:824-831, 1992 and Ferrara et al., Ann. NY Acad.Sci. 732:246-256, 1995), which measures the formation of tube-likestructures by microvascular endothelial cells; and matrigel models(Grant et al., “Angiogenesis as a component of epithelial-mesenchymalinteractions” in Goldberg and Rosen, Epithelial-Mesenchymal Interactionin Cancer, Birkhäuser Verlag, 1995, 235-248; Baatout, AnticancerResearch 17:451-456, 1997), which are used to determine effects on cellmigration and tube formation by endothelial cells seeded in matrigel, abasement membrane extract enriched in laminin. Angiogenesis assays canbe carried out in the presence and absence of VEGF to assess possiblecombinatorial effects. VEGF can be used as a control within in vivoassays.

Zvegf3 activity can also be measured using assays that measure axonguidance and growth. Of particular interest are assays that indicatechanges in neuron growth patterns, for example those disclosed inHastings, WIPO Publication WO 97/29189 and Walter et al., Development101:685-96, 1987. Assays to measure the effects on neuron growth arewell known in the art. For example, the C assay (e.g., Raper andKapfhammer, Neuron 4:21-9, 1990 and Luo et al., Cell 75:217-27, 1993)can be used to determine collapsing activity of zvegf3 on growingneurons. Other methods that can assess zvegf3-induced effects on neuriteextension are also known. See, Goodman, Annu. Rev. Neurosci. 19:341-77,1996. Conditioned media from cells expressing a zvegf3 protein, a zvegf3agonist, or a zvegf3 antagonist, or aggregates of such cells, can byplaced in a gel matrix near suitable neural cells, such as dorsal rootganglia (DRG) or sympathetic ganglia explants, which have beenco-cultured with nerve growth factor. Compared to control cells,zvegf3-induced changes in neuron growth can be measured (as disclosedby, for example, Messersmith et al., Neuron 14:949-59, 1995 and Puschelet al., Neuron 14:941-8, 1995). Likewise neurite outgrowth can bemeasured using neuronal cell suspensions grown in the presence ofmolecules of the present invention. See, for example, O'Shea et al.,Neuron 7:231-7, 1991 and DeFreitas et al., Neuron 15:333-43, 1995.

The biological activities of zvegf3 proteins can be studied in non-humananimals by administration of exogenous protein, by expression ofzvegf3-encoding polynucleotides, and by suppression of endogenous zvegf3expression through antisense or knock-out techniques. Zvegf3 proteinscan be administered or expressed individually, in combination with otherzvegf3 proteins, or in combination with non-vegf3 proteins, includingother growth factors (e.g., other VEGFs, PlGFs, or PDGFs). For example,a combination of zvegf3 polypeptides (e.g., a combination ofzvegf3₁₅₋₁₆₃, zvegf3₁₅₋₂₃₀, and zvegf3₂₃₅₋₃₄₅) can be administered to atest animal or expressed in the animal. Test animals are monitored forchanges in such parameters as clinical signs, body weight, blood cellcounts, clinical chemistry, histopathology, and the like.

Effects of zvegf3 and zvegf3 antagonists on liver and kidney fibrosiscan be tested in known animal models, such as the db/db mouse modeldisclosed by Cohen et al., Diabetologia 39:270-274, 1996 and Cohen etal., J. Clin. Invest. 95:2338-2345, 1995 or transgenic animal models(Imai et al., Contrib. Nephrol. 107:205-215, 1994).

Effects on fibrosis can also be assayed in a mouse model usingbleomycin. The chemotherapy agent bleomycin is a known causative agentof pulmonary fibrosis in humans and can induce interstitial lung diseasein mice, including an increase in the number of fibroblasts, enhancedcollagen deposition, and dysregulated matrix remodeling. C57Bl/6 miceare administered bleomycin by osmotic minipump for 1 week. There followsa period of inflammation, with cutaneous toxicity beginningapproximately 4-7 days after bleomycin administration and continuing forabout a week, after which the mice appear to regain health. About 3-4weeks after the finish of bleomycin delivery, the mice are sacrificed,and the lungs are examined histologically for signs of fibrosis. Scoringis based on the extent of lung fibrotic lesions and their severity.Serum is assayed for lactic dehydrogenase, an intracellular enzyme thatis released into the circulation upon general cell death or injury. Lungtissue is assayed for hydroxyproline as a measure of collagendeposition.

Stimulation of coronary collateral growth can be measured in knownanimal models, including a rabbit model of peripheral limb ischemia andhind limb ischemia and a Pig model of chronic myocardial ischemia(Ferrara et al., Endocrine Reviews 18:4-25, 1997). Zvegf3 proteins areassayed in the presence and absence of VEGFs, angiopoietins, and basicFGF to test for combinatorial effects. These models can be modified bythe use of adenovirus or naked DNA for gene delivery as disclosed inmore detail below, resulting in local expression of the test protein(s).

Efficacy of zvegf3 polypeptides in promoting wound healing can beassayed in animal models. One such model is the linear skin incisionmodel of Mustoe et al. (Science 237:1333, 1987). In a typical procedure,a 6-cm incision is made in the dorsal pelt of an adult rat, then closedwith wound clips. Test substances and controls (in solution, gel, orpowder form) are appied before primary closure. Administration willoften be limited to a single application, although additionalapplications can be made on succeeding days by careful injection atseveral sites under the incision. Wound breaking strength is evaluatedbetween 3 and 21 days post wounding. In a second model, multiple, small,full-thickness excisions are made on the ear of a rabbit. The cartilagein the ear splints the wound, removing the variable of wound contractionfrom the evaluation of closure. Experimental treatments and controls areapplied. The geometry and anatomy of the wound site allow for reliablequantification of cell ingrowth and epithelial migration, as well asquantitative analysis of the biochemistry of the wounds (e.g., collagencontent). See, Mustoe et al., J. Clin. Invest. 87:694, 1991. The rabbitear model can be modified to create an ischemic wound environment, whichmore closely resembles the clinical situation (Ahn et al., Ann. Plast.Surg. 24:17, 1990). Within a third model, healing of partial-thicknessskin wounds in pigs or guinea pigs is evaluated (LeGrand et al., GrowthFactors 8:307, 1993). Experimental treatments are applied daily on orunder dressings. Seven days after wounding, granulation tissue thicknessis determined. This model is particularly useful for dose-responsestudies, as it is more quantitative than other in vivo models of woundhealing. A full thickness excision model can also be employed. Withinthis model, the epidermis and dermis are removed down to the panniculuscarnosum in rodents or the subcutaneous fat in pigs. Experimentaltreatments are applied topically on or under a dressing, and can beapplied daily if desired. The wound closes by a combination ofcontraction and cell ingrowth and proliferation. Measurable endpointsinclude time to wound closure, histologic score, and biochemicalparameters of wound tissue. Impaired wound healing models are also knownin the art (e.g., Cromack et al., Surgery 113:36, 1993; Pierce et al.,Proc. Natl. Acad. Sci. USA 86:2229, 1989; Greenhalgh et al., Amer. J.Pathol. 136:1235, 1990). Delay or prolongation of the wound healingprocess can be induced pharmacologically by treatment with steroids,irradiation of the wound site, or by concomitant disease states (e.g.,diabetes). Linear incisions or full-thickness excisions are mostcommonly used as the experimental wound. Endpoints are as disclosedabove for each type of wound. Subcutaneous implants can be used toassess compounds acting in the early stages of wound healing (Broadleyet al., Lab. Invest. 61:571, 1985; Sprugel et al., Amer. J. Pathol. 129:601, 1987). Implants are prepared in a porous, relativelynon-inflammatory container (e.g., polyethylene sponges or expandedpolytetrafluoroethylene implants filled with bovine collagen) and placedsubcutaneously in mice or rats. The interior of the implant is empty ofcells, producing a “wound space” that is well-defined and separable fromthe preexisting tissue. This arrangement allows the assessment of cellinflux and cell type as well as the measurement ofvasculogenesis/angiogenesis and extracellular matrix production.

Expression of zvegf3 proteins in animals provides models for study ofthe biological effects of overproduction or inhibition of proteinactivity in vivo. Zvegf3-encoding polynucleotides can be introduced intotest animals, such as mice, using viral vectors or naked DNA, ortransgenic animals can be produced. In general, a zvegf3 protein isexpressed with a secretory peptide. Suitable secretory peptides includethe zvegf3 secretory peptide (e.g., residues 1-14 of SEQ ID NO:2) andheterologous secretory peptides. An exemplary heterologous secretorypeptide is that of human tissue plasminogen activator (t-PA). The t-PAsecretory peptide may be modified to reduce undesired proteolyticcleavage as disclosed in U.S. Pat. No. 5,641,655.

Proteins of the present invention can be assayed in vivo using viraldelivery systems. Exemplary viruses include adenovirus, herpesvirus,retroviruses, vaccinia virus, and adeno-associated virus (AAV). Forreview, see Becker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglasand Curiel, Science & Medicine 4:44-53, 1997. Adenovirus (reviewed byBecker et al., Meth. Cell Biol. 43:161-89, 1994; and Douglas and Curiel,Science &Medicine 4:44-53, 1997) offers several advantages. Adenoviruscan (i) accommodate relatively large DNA inserts; (ii) be grown tohigh-titer; (iii) infect a broad range of mammalian cell types; and (iv)be used with many different promoters including ubiquitous, tissuespecific, and regulatable promoters. Because adenoviruses are stable inthe bloodstream, they can be administered by intravenous injection. Ifthe adenoviral delivery system has an E1 gene deletion, the virus cannotreplicate in the host cells. However, the host's tissue (e.g., liver)will express and process (and, if a secretory signal sequence ispresent, secrete) the heterologous protein. Secreted proteins will enterthe circulation in the highly vascularized liver, and effects on theinfected animal can be determined. Adenoviral vectors containing variousdeletions of viral genes can be used in an attempt to reduce oreliminate immune responses to the vector. Such adenoviruses are E1deleted, and in addition contain deletions of E2A or E4 (Lusky et al.,J. Virol. 72:2022-2032, 1998; Raper et al., Human Gene Therapy9:671-679, 1998). In addition, deletion of E2b is reported to reduceimmune responses (Amalfitano, et al., J. Virol. 72:926-933, 1998).Generation of so-called “gutless” adenoviruses where all viraltranscription units are deleted is particularly advantageous forinsertion of large inserts of heterologous DNA. For review, see Yeh andPerricaudet, FASEB J. 11:615-623, 1997. Retroviral vectors aredisclosed, for example, by Anderson et al., U.S. Pat. No. 5,399,346;Mann et al., Cell 33:153, 1983; Temin et al., U.S. Pat. No. 4,650,764;Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al., J. Virol.62:1120, 1988; Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al.,WIPO publication WO 95/07358; and Kuo et al., Blood 82:845, 1993.

In an alternative method, a vector can be introduced byliposome-mediated transfection, a technique that provides certainpractical advantages, including the molecular targeting of liposomes tospecific cells. Directing transfection to particular cell types isparticularly advantageous in a tissue with cellular heterogeneity, suchas the pancreas, liver, kidney, and brain. Lipids may be chemicallycoupled to other molecules for the purpose of targeting. Targetedpeptides (e.g., hormones or neurotransmitters), proteins such asantibodies, or non-peptide molecules can be coupled to liposomeschemically.

Within another embodiment target cells are removed from the the animal,and DNA is introduced as a naked DNA plasmid. The transformed cells arethen re-implanted into the body of the animal. Naked DNA vectors can beintroduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a gene gunor use of a DNA vector transporter. See, e.g., Wu et al., J. Biol. Chem.267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.

Mice engineered to express the zvegf3 gene, referred to as “transgenicmice,” and mice that exhibit a complete absence of zvegf3 gene function,referred to as “knockout mice,” can also be generated (Snouwaert et al.,Science 257:1083, 1992; Lowell et al., Nature 366:740-42, 1993;Capecchi, Science 244:1288-1292, 1989; Palmiter et al., Ann. Rev. Genet.20:465-499, 1986). Transgenesis experiments can be performed usingnormal mice or mice with genetic disease or other altered phenotypes.Transgenic mice that over-express zvegf3, either ubiquitously or under atissue-specific or tissue-restricted promoter, can be used to determinewhether or not over-expression causes a phenotypic change. Suitablepromoters include metallothionein, albumin (Pinkert et al., Genes Dev.1(3):268-76, 1987), and K-14 keratinocyte (Vassar et al., Proc. Natl.Acad. Sci. USA 86(5):1563-1567, 1989) gene promoters. Themetallothionein-1 (MT-1) promoter provides expression in liver and othertissues, often leading to high levels of circulating protein.Over-expression of a wild-type zvegf3 polypeptide, polypeptide fragmentor a mutant thereof may alter normal cellular processes, resulting in aphenotype that identifies a tissue in which zvegf3 expression isfunctionally relevant and may indicate a therapeutic target for thezvegf3, its agonists or antagonists. For example, a transgenic mouse canbe engineered to over-express a full-length zvegf3 sequence, which mayresult in a phenotype that shows similarity with human diseases.Similarly, knockout zvegf3 mice can be used to determine where zvegf3 isabsolutely required in vivo. The phenotype of knockout mice ispredictive of the in vivo effects of zvegf3 antagonists. Knockout micecan also be used to study the effects of zvegf3 proteins in models ofdisease, including, for example, cancer, atherosclerosis, rheumatoidarthritis, ischemia, and cardiovascular disease. The human zvegf3 cDNAcan be used to isolate murine zvegf3 mRNA, cDNA and genomic DNA asdisclosed above, which are subsequently used to generate knockout mice.These mice may be employed to study the zvegf3 gene and the proteinencoded thereby in an in vivo system, and can be used as in vivo modelsfor corresponding human diseases. Moreover, transgenic mice expressingzvegf3 antisense polynucleotides or ribozymes directed against zvegf3,described herein, can be used analogously to knockout mice describedabove.

The functional relationship between zvegf3 and PDGF ligands can beinvestigated through knockin experiments. For example, the PDGF A orPDGF B gene in an animal can be replaced by knocking a zvegf3 gene orcDNA into the genomic locus of a PDGF ligand in embryonic stem (ES)cells, which are then used to generate the knockin mice, in which zvegf3protein is expressed by the (replaced) PDGF genomic locus. Such knockinmice can be used to address a number of questions, such as whetherzvegf3 can substitute PDGF function during development; whether zvegf3has unique functions; and, using cell lines established from the mice,the mechanism of zvegf3 signal transduction. See, for example, Wang etal., Development 124:2507-2513, 1997; Zhuang et al., Mol. Cell Biol.18:3340-3349. 1998; Geng et al., Cell 97:767-777, 1999. In a furtherapplication of this technology, individual domains of zvegf3 can bespecifically deleted or modified by knocking in a modified zvegf3sequence or a specific spliced version of zvegf3, thereby providing atransgenic model for the study of functional domains of the protein.See, for example, Zhuang et al. (ibid.) and Baudoin et al. (Genes Dev.12:1202-1216, 1998). In another application, tissues and cell types thatexpress the zvegf3 gene can be identified by knockin of a sensitivereporter gene (e.g., LacZ) into the zvegf3 locus. See, for example,Monroe et al., Immunity 11:201-212, 1999; Zhuang et al., ibid.; Geng etal., ibid.

Antisense methodology can be used to inhibit zvegf3 gene transcriptionto examine the effects of such inhibition in vivo. Polynucleotides thatare complementary to a segment of a zvegf3-encoding polynucleotide(e.g., a polynucleotide as set froth in SEQ ID NO:1) are designed tobind to zvegf3-encoding mRNA and to inhibit translation of such mRNA.Such antisense oligonucleotides can also be used to inhibit expressionof zvegf3 polypeptide-encoding genes in cell culture.

Those skilled in the art will recognize that the assays disclosed hereincan be readily adapted to study the activity of zvegf3 proteins,anti-zvegf3 antibodies and other antagonists, and test substancesderived from a variety of sources.

Zvegf3 proteins may be used therapeutically to stimulate tissuedevelopment or repair, or cellular differentiation or proliferation.Zvegf3 has been found to bind to PDGF alpha receptor and to stimulatealpha receptor-mediated cellular processes. The protein can therefore beused as a PDGF alpha receptor agonist. Specific applications include,without limitation: the treatment of full-thickness skin wounds,including venous stasis ulcers and other chronic, non-healing wounds,particularly in cases of compromised wound healing due to diabetesmellitus, connective tissue disease, smoking, burns, and otherexacerbating conditions; fracture repair; skin grafting; withinreconstructive surgery to promote neovascularization and increase skinflap survival; to establish vascular networks in transplanted cells andtissues, such as transplanted islets of Langerhans; to treat femalereproductive tract disorders, including acute or chronic placentalinsufficiency (an important factor causing perinatal morbidity andmortality) and prolonged bleeeding; to promote the growth of tissuedamaged by periodontal disease; to promote the repair of damaged livertissue; in the treatment of acute and chronic lesions of thegastrointestinal tract, including duodenal ulcers, which arecharacterized by a deficiency of microvessels; to promote angiogenesisand prevent neuronal degeneration due to chronic cerebral ischemia; toaccelerate the formation of collateral blood vessels in ischemic limbs;to promote vessel repair and development of collateral circulationfollowing myocardial infarction so as to limit ischemic injury; and tostimulate hematopoiesis. The polypeptides are also useful additives intissue adhesives for promoting revascularization of the healing tissue.

Of particular interest is the use of zvegf3 or zvegf3 antagonists forthe treatment or repair of liver damage, including damage due to chronicliver disease, including chronic active hepatitis (including hepatitisC) and many other types of cirrhosis. Widespread, massive necrosis,including destruction of virtually the entire liver, can be caused by,inter alia, fulminant viral hepatitis; overdoses of the analgesicacetaminophen; exposure to other drugs and chemicals such as halothane,monoamine oxidase inhibitors, agents employed in the treatment oftuberculosis, phosphorus, carbon tetrachloride, and other industrialchemicals. Conditions associated with ultrastructural lesions that donot necessarily produce obvious liver cell necrosis include Reye'ssyndrome in children, tetracycline toxicity, and acute fatty liver ofpregnancy. Cirrhosis, a diffuse process characterized by fibrosis and aconversion of normal architecture into structurally abnormal nodules,can come about for a variety reasons including alcohol abuse, postnecrotic cirrhosis (usually due to chronic active hepatitis), biliarycirrhosis, pigment cirrhosis, cryptogenic cirrhosis, Wilson's disease,and alpha-1-antitrypsin deficiency. Zvegf3 may also be useful for thetreatment of hepatic chronic passive congestion (CPC) and centralhemorrhagic necrosis (CHN), which are two circulatory changesrepresenting a continuum encountered in right-sided heart failure. Othercirculatory disorders that may be treated with zvegf3 include hepaticvein thrombosis, portal vein thrombosis, and cardiac sclerosis. In casesof liver fibrosis it may be beneficial to administer a zvegf3 antagonistto suppress the activation of stellate cells, which have been implicatedin the production of extracellular matrix in fibrotic liver (Li andFriedman, J. Gastroenterol. Hepatol. 14:618-633, 1999).

Zvegf3 polypeptides can be administered alone or in combination withother vasculogenic or angiogenic agents, including VEGF. For example,basic and acidic FGFs and VEGF have been found to play a role in thedevelopment of collateral circulation, and the combined use of zvegf3with one or more of these factors may be advantageous. VEGF has alsobeen implicated in the survival of transplanted islet cells (Gorden etal. Transplantation 63:436-443, 1997; Pepper, Arteriosclerosis, Throm.and Vascular Biol. 17:605-619, 1997). Basic FGF has been shown to induceangiogenesis and accelerate healing of ulcers in experimental animals(reviewed by Folkman, Nature Medicine 1:27-31, 1995). VEGF has beenshown to promote vessel re-endothelialization and to reduce intimalhyperplasia in animal models of restenosis (Asahara et al., Circulation91:2802-2809, 1995; Callow et al., Growth Factors 10:223-228, 1994);efficacy of zvegf3 polypeptides can be tested in these and other knownmodels. When using zvegf3 in combination with an additional agent, thetwo compounds can be administered simultaneously or sequentially asappropriate for the specific condition being treated.

Zvegf3 proteins may be used either alone or in combination with otherhematopoietic factors such as IL-3, G-CSF, GM-CSF, or stem cell factorto enhance expansion and mobilization of endothelial precursor stemcells. Cells that can be expanded in this manner include cells isolatedfrom bone marrow or cells isolated from blood. Zvegf3 proteins may alsobe given directly to an individual to enhance endothelial stem cellproduction and differentiation within the treated individual. The stemcells, either developed within the patient, or provided back to apatient, may then play a role in modulating areas of ischemia within thebody, thereby providing a therapeutic effect. These cells may also beuseful in enhancing re-endothelialization of areas devoid of endothelialcoverage, such as vascular grafts, vascular stents, and areas where theendothelial coverage has been damaged or removed (e.g., areas ofangioplasty). Zvegf3 proteins may also be used in combination with othergrowth and differentiation factors such as angiopoietin-1 (Davis et al.,Cell 87:1161-1169, 1996) to help create and stabilize new vesselformation in areas requiring neovascularization, including areas ofischemia (cardiac or peripheral ischemia), organ transplants, woundhealing, and tissue grafting.

Zvegf3 proteins, agonists and antagonists may be used to modulateneurite growth and development and demarcate nervous system structures.As such, Zvegf3 proteins, agonists, or antagonists may useful in thetreatment of peripheral neuropathies by increasing spinal cord andsensory neurite outgrowth, and as part of a therapeutic treatment forthe regeneration of neurite outgrowths following strokes, brain damagecaused by head injuries, and paralysis caused by spinal injuries.Application may also be made in treating neurodegenerative diseases suchas multiple sclerosis, Alzheimer's disease and Parkinson's disease.Application may also be made in mediating development and innervationpattern of stomach tissue.

For pharmaceutical use, zvegf3 proteins are formulated for topical orparenteral, particularly intravenous or subcutaneous, delivery accordingto conventional methods. In general, pharmaceutical formulations willinclude a zvegf3 polypeptide in combination with a pharmaceuticallyacceptable vehicle, such as saline, buffered saline, 5% dextrose inwater, or the like. Formulations may further include one or moreexcipients, preservatives, solubilizers, buffering agents, albumin toprevent protein loss on vial surfaces, etc. Methods of formulation arewell known in the art and are disclosed, for example, in Remington: TheScience and Practice of Pharmacy, Gennaro, ed., Mack Publishing Co.,Easton, Pa, 19th ed., 1995. Zvegf3 will ordinarily be used in aconcentration of about 10 to 100 μg/ml of total volume, althoughconcentrations in the range of 1 ng/ml to 1000 μg/ml may be used. Fortopical application, such as for the promotion of wound healing, theprotein will be applied in the range of 0.1-10 μg/cm² of wound area,with the exact dose determined by the clinician according to acceptedstandards, taking into account the nature and severity of the conditionto be treated, patient traits, etc. Determination of dose is within thelevel of ordinary skill in the art. The therapeutic formulations willgenerally be administered over the period required forneovascularization, typically from one to several months and, intreatment of chronic conditions, for a year or more. Dosing is daily orintermittently over the period of treatment. Intravenous administrationwill be by bolus injection or infusion over a typical period of one toseveral hours. Sustained release formulations can also be employed. Ingeneral, a therapeutically effective amount of zvegf3 is an amountsufficient to produce a clinically significant change in the treatedcondition, such as a clinically significant reduction in time requiredby wound closure, a significant reduction in wound area, a significantimprovement in vascularization, a significant reduction in morbidity, ora significantly increased histological score.

Proteins of the present invention are useful for modulating theproliferation, differentiation, migration, or metabolism of responsivecell types, which include both primary cells and cultured cell lines. Ofparticular interest in this regard are liver cells, hematopoietic cells(including stem cells and mature myeloid and lymphoid cells),endothelial cells, neuronal cells, and mesenchymal cells (includingfibroblasts and smooth muscle cells). Zvegf3 polypeptides are added totissue culture media for these cell types at a concentration of about 10pg/ml to about 1000 ng/ml. Those skilled in the art will recognize thatzvegf3 proteins can be advantageously combined with other growth factorsin culture media.

Within the laboratory research field, zvegf3 proteins can also be usedas molecular weight standards; as reagents in assays for determiningcirculating levels of the protein, such as in the diagnosis of disorderscharacterized by over- or under-production of zvegf3 protein; or asstandards in the analysis of cell phenotype.

Zvegf3 proteins can also be used to identify inhibitors of theiractivity. Test compounds are added to the assays disclosed above toidentify compounds that inhibit the activity of zvegf3 protein. Inaddition to those assays disclosed above, samples can be tested forinhibition of zvegf3 activity within a variety of assays designed tomeasure receptor binding or the stimulation/inhibition ofzvegf3-dependent cellular responses. For example, zvegf3-responsive celllines can be transfected with a reporter gene construct that isresponsive to a zvegf3-stimulated cellular pathway. Reporter geneconstructs of this type are known in the art, and will generallycomprise a zvegf3-activated serum response element (SRE) operably linkedto a gene encoding an assayable protein, such as luciferase. Candidatecompounds, solutions, mixtures or extracts are tested for the ability toinhibit the activity of zvegf3 on the target cells as evidenced by adecrease in zvegf3 stimulation of reporter gene expression. Assays ofthis type will detect compounds that directly block zvegf3 binding tocell-surface receptors, as well as compounds that block processes in thecellular pathway subsequent to receptor-ligand binding. In thealternative, compounds or other samples can be tested for directblocking of zvegf3 binding to receptor using zvegf3 tagged with adetectable label (e.g., ¹²⁵I, biotin, horseradish peroxidase, FITC, andthe like). Within assays of this type, the ability of a test sample toinhibit the binding of labeled zvegf3 to the receptor is indicative ofinhibitory activity, which can be confirmed through secondary assays.Receptors used within binding assays may be cellular receptors orisolated, immobilized receptors.

The activity of zvegf3 proteins can be measured with a silicon-basedbiosensor microphysiometer that measures the extracellular acidificationrate or proton excretion associated with receptor binding and subsequentphysiologic cellular responses. An exemplary such device is theCytosensor™ Microphysiometer manufactured by Molecular Devices,Sunnyvale, Calif. A variety of cellular responses, such as cellproliferation, ion transport, energy production, inflammatory response,regulatory and receptor activation, and the like, can be measured bythis method. See, for example, McConnell et al., Science 257:1906-1912,1992; Pitchford et al., Meth. Enzymol. 228:84-108, 1997; Arimilli etal., J. Immunol. Meth. 212:49-59, 1998; and Van Liefde et al., Eur. J.Pharmacol. 346:87-95, 1998. The microphysiometer can be used forassaying adherent or non-adherent eukaryotic or prokaryotic cells. Bymeasuring extracellular acidification changes in cell media over time,the microphysiometer directly measures cellular responses to variousstimuli, including zvegf3 proteins, their agonists, and antagonists. Themicrophysiometer can be used to measure responses of a zvegf3-responsiveeukaryotic cell, compared to a control eukaryotic cell that does notrespond to zvegf3 polypeptide. Zvegf3-responsive eukaryotic cellscomprise cells into which a receptor for zvegf3 has been transfectedcreating a cell that is responsive to zvegf3, as well as cells naturallyresponsive to zvegf3 such as cells derived from vascular or neuraltissue. Differences, measured by a change, for example, an increase ordiminution in extracellular acidification, in the response of cellsexposed to zvegf3 polypeptide, relative to a control not exposed tozvegf3, are a direct measurement of zvegf3-modulated cellular responses.Moreover, such zvegf3-modulated responses can be assayed under a varietyof stimuli. The present invention thus provides methods of identifyingagonists and antagonists of zvegf3 proteins, comprising providing cellsresponsive to a zvegf3 polypeptide, culturing a first portion of thecells in the absence of a test compound, culturing a second portion ofthe cells in the presence of a test compound, and detecting a change,for example, an increase or diminution, in a cellular response of thesecond portion of the cells as compared to the first portion of thecells. The change in cellular response is shown as a measurable changein extracellular acidification rate. Culturing a third portion of thecells in the presence of a zvegf3 protein and the absence of a testcompound provides a positive control for the zvegf3-responsive cells anda control to compare the agonist activity of a test compound with thatof the zvegf3 polypeptide. Antagonists of zvegf3 can be identified byexposing the cells to zvegf3 protein in the presence and absence of thetest compound, whereby a reduction in zvegf3-stimulated activity isindicative of antagonist activity in the test compound.

Zvegf3 proteins can also be used to identify cells, tissues, or celllines that respond to a zvegf3-stimulated pathway. The microphysiometer,described above, can be used to rapidly identify ligand-responsivecells, such as cells responsive to zvegf3 proteins. Cells are culturedin the presence or absence of zvegf3 polypeptide. Those cells thatelicit a measurable change in extracellular acidification in thepresence of zvegf3 are responsive to zvegf3. Responsive cells can thanbe used to identify antagonists and agonists of zvegf3 polypeptide asdescribed above.

Inhibitors of zvegf3 activity (zvegf3 antagonists) include anti-zvegf3antibodies, soluble zvegf3 receptors (including soluble PDGF alphareceptor; see, e.g., Herren et al., J. Biol. Chem. 268:15088-15095,1993), anti-receptor antibodies, and other peptidic and non-peptidicagents, including ribozymes, small molecule inhibitors, andangiogenically or mitogenically inactive receptor-binding fragments ofzvegf3 polypeptides. Such antagonists can be use to block the mitogenic,chemotactic, or angiogenic effects of zvegf3. These antagonists maytherefore be useful in reducing the growth of solid tumors by inhibitingneovascularization of the developing tumor, by directly blocking tumorcell growth, or by promoting apoptosis through the inhibition of PDGFalpha receptor-mediated processes. For example, experimental evidenceindicates that zvegf3 is produced by glioblastoma cells, suggesting thatinhibition of zvegf3 activity may be useful in the treatment of thesetumors. Other uses of zvegf3 antagonists include treating diabeticretinopathy, psoriasis, arthritis, and scleroderma; and reducingfibrosis, including scar formation, keloids, liver fibrosis, lungfibrosis (e.g., silicosis, asbestosis), kidney fibrosis (includingdiabetic nephropathy), and glomerulosclerosis. Inhibitors of zvegf3 mayalso be useful in the treatment of proliferative vascular disorderswherein zvegf3 activity is pathogenic. Such disorders may includeatherosclerosis and intimal hyperplastic restenosis followingangioplasty, endarterectomy, vascular grafting, organ transplant, orvascular stent emplacement. These conditions involve complex growthfactor-mediated responses wherein certain factors may be beneficial tothe clinical outcome and others may be pathogenic.

Inhibitors of zvegf3 may also prove useful in the treatment of ocularneovascularization, including diabetic retinopathy and age-relatedmacular degeneration. Experimental evidence suggests that theseconditions result from the expression of angiogenic factors induced byhypoxia in the retina.

Zvegf3 antagonists are also of interest in the treatment of inflammatorydisorders, such as rheumatoid arthritis and psoriasis. In rheumatoidarthritis, studies suggest that VEGF plays an important role in theformation of pannus, an extensively vascularized tissue that invades anddestroys cartilage. Psoriatic lesions are hypervascular and overexpressthe angiogenic polypeptide IL-8.

Zvegf3 antagonists may also prove useful in the treatment of infantilehemangiomas, which exhibit overexpression of VEGF and bFGF during theproliferative phase.

Inhibitors are formulated for pharmaceutical use as generally disclosedabove, taking into account the precise chemical and physical nature ofthe inhibitor and the condition to be treated. The relevantdeterminations are within the level of ordinary skill in the formulationart. Other angiogenic and vasculogenic factors, including VEGF and bFGF,have been implicated in pathological neovascularization. In suchinstances it may be advantageous to combine a zvegf3 inhibitor with oneor more inhibitors of these other factors.

The polypeptides, nucleic acids, and antibodies of the present inventionmay be used in diagnosis or treatment of disorders associated with cellloss or abnormal cell proliferation, including cancer, impaired orexcessive vasculogenesis or angiogenesis, and diseases of the nervoussystem. Labeled zvegf3 polypeptides may be used for imaging tumors orother sites of abnormal cell proliferation. In view of the binding ofzvegf3 to the PDGF alpha receptor, zvegf3 antagonists may be useful inthe treatment of tumors that express this receptor (e.g., glioblastomas,malignant melanomas). Because angiogenesis in adult animals is generallylimited to wound healing and the female reproductive cycle, it is a veryspecific indicator of pathological processes. Angiogenesis is indicativeof, for example, developing solid tumors, retinopathies, and arthritis.

Zvegf3 polypeptides and anti-zvegf3 antibodies can be directly orindirectly conjugated to drugs, toxins, radionuclides and the like, andthese conjugates used for in vivo diagnostic or therapeuticapplications. For instance, polypeptides or antibodies of the presentinvention may used to identify or treat tissues or organs that express acorresponding anti-complementary molecule (receptor or antigen,respectively, for instance). More specifically, zvegf3 polypeptides oranti-zvegf3 antibodies, or bioactive fragments or portions thereof, canbe coupled to detectable or cytotoxic molecules and delivered to amammal having cells, tissues, or organs that express theanti-complementary molecule. For example, the CUB domain of zvegf3 canbe used to target peptidic and non-peptidic moieties to semaphorins asdisclosed above.

Suitable detectable molecules can be directly or indirectly attached tothe polypeptide or antibody, and include radionuclides, enzymes,substrates, cofactors, inhibitors, fluorescent markers, chemiluminescentmarkers, magnetic particles, and the like. Suitable cytotoxic moleculescan be directly or indirectly attached to the polypeptide or antibody,and include bacterial or plant toxins (for instance, diphtheria toxin,Pseudomonas exotoxin, ricin, abrin, saporin, and the like), as well astherapeutic radionuclides, such as iodine-131, rhenium-188 oryttrium-90. These can be either directly attached to the polypeptide orantibody, or indirectly attached according to known methods, such asthrough a chelating moiety. Polypeptides or antibodies can also beconjugated to cytotoxic drugs, such as adriamycin. For indirectattachment of a detectable or cytotoxic molecule, the detectable orcytotoxic molecule may be conjugated with a member of acomplementary/anticomplementary pair, where the other member is bound tothe polypeptide or antibody portion. For these purposes,biotin/streptavidin is an exemplary complementary/anticomplementarypair.

In another embodiment, polypeptide-toxin fusion proteins orantibody/fragment-toxin fusion proteins may be used for targeted cell ortissue inhibition or ablation, such as in cancer therapy. Of particularinterest in this regard are conjugates of a zvegf3 polypeptide and acytotoxin, which can be used to target the cytotoxin to a tumor or othertissue that is undergoing undesired cell replication and modification.

In another embodiment, zvegf3-cytokine fusion proteins orantibody/fragment-cytokine fusion proteins may be used for enhancing invitro cytotoxicity (for instance, that mediated by monoclonal antibodiesagainst tumor targets) and for enhancing in vivo killing of targettissues (for example, blood and bone marrow cancers). See, generally,Hornick et al., Blood 89:4437-4447, 1997). In general, cytokines aretoxic if administered systemically. The described fusion proteins enabletargeting of a cytokine to a desired site of action, such as a cellhaving binding sites for zvegf3, thereby providing an elevated localconcentration of cytokine. Suitable cytokines for this purpose include,for example, interleukin-2 and granulocyte-macrophage colony-stimulatingfactor (GM-CSF). Such fusion proteins may be used to causecytokine-induced killing of tumors and other tissues exhibitingundesired cell replication.

In yet another embodiment, a zvegf3 polypeptide or anti-zvegf3 antibodycan be conjugated with a radionuclide, particularly with a beta-emittingor gamma-emitting radionuclide, and used to reduce restenosis. Forinstance, iridium-192 impregnated ribbons placed into stented vessels ofpatients until the required radiation dose was delivered resulted indecreased tissue growth in the vessel and greater luminal diameter thanthe control group, which received placebo ribbons. Further,revascularisation and stent thrombosis were significantly lower in thetreatment group. Similar results are predicted with targeting of abioactive conjugate containing a radionuclide, as described herein.

The bioactive polypeptide or antibody conjugates described herein can bedelivered intravenously, intra-arterially or intraductally, or may beintroduced locally at the intended site of action.

Polynucleotides encoding zvegf3 polypeptides are useful within genetherapy applications where it is desired to increase or inhibit zvegf3activity. For example, Isner et al., The Lancet (ibid.) reported thatVEGF gene therapy promoted blood vessel growth in an ischemic limb.Additional applications of zvegf3 gene therapy include stimulation ofwound healing, repopulation of vascular grafts, stimulation of neuritegrowth, and inhibition of cancer growth and metastasis.

The present invention also provides polynucleotide reagents fordiagnostic use. For example, a zvegf3 gene, a probe comprising zvegf3DNA or RNA, or a subsequence thereof can be used to determine if thezvegf3 gene is present on chromosome 4 of a human patient or if amutation has occurred. Detectable chromosomal aberrations at the zvegf3gene locus include, but are not limited to, aneuploidy, gene copy numberchanges, insertions, deletions, restriction site changes andrearrangements. Such aberrations can be detected using polynucleotidesof the present invention by employing molecular genetic techniques, suchas restriction fragment length polymorphism (RFLP) analysis, shorttandem repeat (STR) analysis employing PCR techniques, and other geneticlinkage analysis techniques known in the art (Sambrook et al., ibid.;Ausubel et. al., ibid.; A. J. Marian, Chest 108:255-265, 1995).

Radiation hybrid mapping is a somatic cell genetic technique developedfor constructing high-resolution, contiguous maps of mammalianchromosomes (Cox et al., Science 250:245-250, 1990). Partial or fullknowledge of a gene's sequence allows one to design PCR primers suitablefor use with chromosomal radiation hybrid mapping panels. Commerciallyavailable radiation hybrid mapping panels that cover the entire humangenome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel(Research Genetics, Inc., Huntsville, Ala.), are available. These panelsenable rapid, PCR-based chromosomal localizations and ordering of genes,sequence-tagged sites (STSs), and other nonpolymorphic and polymorphicmarkers within a region of interest. This technique allows one toestablish directly proportional physical distances between newlydiscovered genes of interest and previously mapped markers. The preciseknowledge of a gene's position can be useful for a number of purposes,including: 1) determining relationships between short sequences andobtaining additional surrounding genetic sequences in various forms,such as YACs, BACs or cDNA clones; 2) providing a possible candidategene for an inheritable disease which shows linkage to the samechromosomal region; and 3) cross-referencing model organisms, such asmouse, which may aid in determining what function a particular genemight have.

The invention is further illustrated by the following non-limitingexamples.

EXAMPLES Example 1

Clones comprising portions of the zvegf3 coding sequence were identifiedin public and proprietary databases of expressed sequence tags (ESTs). Afirst clone, corresponding to an EST in a proprietary database, wasobtained and sequenced. It contained a 2350-bp insert with an openreading frame of about 800 bp. The 5′ end of the ORF was missing. Asecond clone, corresponding to an EST in a public database, was thensequenced, but it didn't extend the sequence obtained from the firstclone. A third clone, from a proprietary database, was then sequenced.This clone contained approximately 156 bp more than the first clone, butwas also missing the 5′ end.

Northerns were performed using a series of Northern blots (MultipleTissue Northern Blots, Clontech Laboratories, Inc., Palo Alto, Calif.).An approximately 400-bp DNA probe, based directly on the identified EST,was generated by digestion of a clone corresponding to the firstproprietary EST with EcoRI and BglII. The DNA probe was gel-purifiedusing a spin column containing a silica gel membrane (QIAquick™ GelExtraction Kit; Qiagen, Inc., Valencia, Calif.). The probe wasradioactively labeled with ³²P using a commercially availablerandom-prime labeling kit (Rediprime™ II, Amersham Corp., ArlingtonHeights, Ill.) according to the manufacturer's specifications. The probewas purified using a push column (NucTrap® column; Stratagene, La Jolla,Calif.; see U.S. Pat. No. 5,336,412). A commercially availablehybridization solution (ExpressHyb™ Hybridization Solution; ClontechLaboratories, Inc., Palo Alto, Calif.) was used for prehybridization andas a hybridizing solution for the Northern blots. Hybridization tookplace overnight at 65° C., and the blots were then washed 4 times in2×SSC and 0.1% SDS at room temperature, followed by two washes in0.1×SSC and 0.1% SDS at 50° C. and one wash in 0.1×SSC and 0.1% SDS at56° C. The Northern blots were then exposed to film overnight at −80° C.and for three days at −80° C. One transcript size was seen in alltissues at approximately 4.0 kb. Signal intensity was highest inthyroid, spinal cord and adrenal gland. Signal intensity was average inheart, kidney, pancreas, prostate, ovary, stomach, and trachea. Signalintensity was weak in all other tissues.

Northern blot analysis was performed using Mouse Multiple Tissue Blotsobtained from Clontech Laboratories and Invitrogen (Carlsbad, Calif.).An approximately 400 bp DNA probe, based directly on the identified EST,was generated by digestion of a clone corresponding to the firstproprietary EST with EcoRI and BglII. The DNA probe was gel purifiedusing a spin column as disclosed above. The probe was radioactivelylabeled with ³²P by random priming as disclosed above. The probe waspurified using a NucTrap® push column. Hybridization conditions were asdisclosed above. The blots were washed four times in 2×SSC, 0.1% SDS,then twice in 1×SSC at 50° C., then exposed to film overnight at −80° C.and for three days at −80° C. One transcript size of approximately 3.0kb was seen in 7-day embryo. Three-day exposure showed bands of lessintensity in 11-, 15-, and 17-day embryo. The intensity decreased withthe age of the embryo. Dot blots showed a spot in 17-day embryo, andwith-three day exposure in submaxillary gland. A potential band ofapproximately 2.0 kb was seen in testis after three days.

Southern blot analysis was performed using a pre-made Southern blotcontaining EcoRI-digested genomic DNA from nine different eukaryoticspecies (ZOO-BLOT from Clontech Laboratories). An approximately 400 bpDNA probe, based directly on the first proprietary database EST, wasgenerated by digestion of the corresponding clone with EcoRI and BglII.The DNA probe was gel purified using a spin column. The probe wasradioactively labeled with ³²P by random priming and purified using apush column. Hybridization and wash conditions were as disclosed abovefor the Mouse Multiple Tissue Blots. The Northern blots were thenexposed to film overnight at −80° C. and for three days at −80° C.Strong bands were seen in rabbit, mouse, rat, and monkey.

Example 2

A human salivary gland library was screened for a full-length clone ofzvegf3 by PCR. This library was an arrayed library representing 9.6×10⁵clones made in the vector pZP5x. The vector pZP5x is the same as vectorpZP-9 (deposited with American Type Culture Collection, 10801 UniversityBlvd., Manassas, Va. under Accession Number 98668), but contains acytomegalovirus promoter instead of a metallothionein promoter betweenthe Asp7l8 and BamHI sites. The plasmid thus comprises a dihyrofolatereductase gene under control of the SV40 early promoter and SV40polyadenylation site, and a cloning site to insert the gene of interestunder control of the CMV promoter and the human growth hormone (hGH)gene polyadenylation site. The working plate containing 80 pools of12,000 colonies each was screened by PCR using oligonucleotide primersZC19,045 (SEQ ID NO:25) and ZC19,047 (SEQ ID NO:26) with an annealingtemperature of 60° C. for 35 cycles. There were two strong positives,pools 58 (T-8 F1-F12) and 77 (T-7 H1-H12). The corresponding pools inthe transfer plate were then screened by PCR using the same conditions.Two positives were obtained at the transfer level. The positives wereT-7 H11 and T-8 F10. 5′ RACE reactions were done on the transfer platepools, and the fragments were sequenced to check zvegf3 sequence anddetermine if a full-length clone was present. For PCR, oligonucleotideprimers ZC12,700 (SEQ ID NO:27) and ZC19,045 (SEQ ID NO:25) were used atan annealing temperature of 61° C. for 5 cycles, then 55° C. for 30cycles. Sequencing showed that the pool T-7 H11 had a frameshift.Transfer plate 8 pool F10 sequence appeared to be correct, so this poolof DNA was used in filter lifts.

Pool F10 from transfer plate 8 was plated and filter lifted using nylonmembranes (Hybond-N™; Amersham Corporation). Approximately 1200 coloniesper plate on each of 5 filters were lifted for a total of approximately6000 colonies. The filters were marked with a hot needle fororientation, then denatured for 6 minutes in 0.5 M NaOH and 1.5 MTris-HCl, pH 7.2. The filters were then neutralized in 1.5 M NaCl and0.5 M Tris-HCl, pH 7.2 for 6 minutes. The DNA was affixed to the filtersusing a UV crosslinker (Stratalinker®, Stratagene, La Jolla, Calif.) at1200 joules. The filters were prewashed at 65° C. in prewash bufferconsisting of 0.25×SSC, 0.25% SDS, and 1 mM EDTA. The solution waschanged a total of three times over a 45-minute period to remove celldebris. Filters were prehybridized for approximately 3 hours at 65° C.in 25 ml of ExpressHyb™. The probe was generated using an approximately400-bp fragment produced by digestion of the first proprietary databaseclone with EcoRI and BglII and gel-purified using a spin column asdisclosed above. The probe was radioactively labeled with ³²P by randompriming as disclosed above and purified using a push column. ExpressHyb™solution was used for the hybridizing solution for the filters.Hybridization took place overnight at 65° C. Blots were rinsed 2× in 65°C. solution 1 (2×SSC, 0.1% SDS), then washed 4 times in solution 1 at65° C. The filters were exposed to film overnight at −80° C. There were14 positives on the filters. 85 clones were picked from the positiveareas and screened by PCR using oligonucleotide primers ZC19,045 (SEQ IDNO:25) and ZC19,047 (SEQ ID NO:26) and an annealing temperature of 60°C. Thirteen positives were obtained and streaked out for individualclones. Twenty-four colonies were picked and checked by PCR aspreviously described. Six positives were obtained, two of which weresequenced. Both sequences were the same and full length. The sequence isshown in SEQ ID NO:1.

Example 3

The human zvegf3 gene was mapped to chromosome 4 using the commerciallyavailable GeneBridge 4 Radiation Hybrid Panel (Research Genetics, Inc.,Huntsville, Ala.). The GeneBridge 4 Radiation Hybrid Panel containsPCRable DNAs from each of 93 radiation hybrid clones, plus two controlDNAs (the HFL donor and the A23 recipient). A publicly available wwwserver (http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapper.pl) allowsmapping relative to the Whitehead Institute/MIT Center for GenomeResearch's radiation hybrid map of the human genome (the “WICGR”radiation hybrid map), which was constructed with the GeneBridge 4Radiation Hybrid Panel.

For the mapping of Zvegf3 with the GeneBridge 4 RH Panel, 20-μlreactions were set up in a PCRable 96-well microtiter plate (Stratagene,La Jolla, Calif.) and used in a thermal cycler (RoboCycler® Gradient 96;Stratagene). Each of the 95 PCR reactions contained 2 μl 10×PCR reactionbuffer (Clontech Laboratories, Inc., Palo Alto, Calif.), 1.6 μl dNTPsmix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 μl sense primerZC 20,368 (SEQ ID NO:28), 1 μl antisense primer ZC 20,369 (SEQ IDNO:29), 2 μl of a density increasing agent and tracking dye (RediLoad,Research Genetics, Inc., Huntsville, Ala.), 0.4 μl of a commerciallyavailable DNA polymerase/antibody mix (50×Advantage™ KlenTaq PolymeraseMix, obtained from Clontech Laboratories, Inc.), 25 ng of DNA from anindividual hybrid clone or control, and x μl ddH₂O for a total volume of20 μl. The reaction mixtures were overlaid with an equal amount ofmineral oil and sealed. The PCR cycler conditions were as follows: aninitial 4-minute denaturation at 94° C.; 35 cycles of 45 secondsdenaturation at 94° C., 45 seconds annealing at 56° C., and 75 secondsextension at 72° C.; followed by a final extension of 7 minutes at 72°C. The reaction products were separated by electrophoresis on a 2%agarose gel (Life Technologies, Gaithersburg, Md.).

The results showed that zvegf3 maps 3.56 cR_(—)3000 from the frameworkmarker CHLC.GATA72A08 on the chromosome 4 WICGR radiation hybrid map.Proximal and distal framework markers were CHLC.GATA72A08 and WI-3936,respectively. The use of surrounding markers positions the zvegf3 genein the 4q28.3 region on the integrated LDB chromosome 4 map (The GeneticLocation Database, University of Southhampton, WWW server:http://cedar.genetics. soton.ac.uk/public_html/).

Using substantially the same methods, the mouse zvegf3 gene was mappedto chromosome 3, linked to the framework marker D3Mit2l2 located at 39.7cM. This region is syntenic with the human zvegf3 locus.

Example 4

A PCR panel was screened for mouse zvegf3 DNA. The panel contained 8cDNA samples from brain, bone marrow, 15-day embryo, testis, salivarygland, placenta, 15-day embryo (Clontech Laboratories), and 17-dayembryo (Clontech Laboratories) libraries.

PCR mixtures contained oligonucleotide primers zc21,222 (SEQ ID NO:38)and zc21,224 (SEQ ID NO:39). The reaction was run at an annealingtemperature of 66° C. with an extension time of 2 minutes for a total of35 cycles using Ex Taq™ DNA polymerase (PanVera, Madison, Wis.) plusantibody. DNA samples found to be positive for zvegf3 by PCR andconfirmed by sequencing included mouse 15-day embryo library total poolcDNA, mouse 15-day embryo (Clontech Laboratories) and 17-day embryo(both obtained from Clontech Laboratories), mouse salivary gland librarytotal pool cDNA, and mouse testis library total pool cDNA. Fragments ofabout 600 bp from each of the mouse 15-day embryo library total poolcDNA, mouse 15-day embryo mcDNA, and mouse 17-day embryo mcDNA PCRproducts were sequenced. Sequence from the mouse 17-day embryo mcDNA andmouse 15-day embryo library total pool cDNA products confirmed thefragments to be mouse zvegf3 DNA.

The mouse 15-day embryo library was screened for full-length zvegf3 DNA.This library was an arrayed library representing 9.6×10⁵ clones in thePCMV.SPORT 2 vector (Life Technologies, Gaithersburg, Md.). The workingplate, containing 80 pools of 12,000 colonies each, was screened by PCRusing oligonucleotide primers zc21,223 (SEQ ID NO:40) and zc21,224 (SEQID NO:39) with an annealing temperature of 66° C. for 35 cycles.Eighteen positives were obtained. Fragments from four pools (A2, A10,B2, and C4)were sequenced; all were confirmed to encode zvegf 3.Additional rounds of screening using the same reaction conditions andpools from the working and source plates identified one positive pool(5D).

Positive colonies were screened by hybridization. Pool 5D from originalsource plate #5 was plated at about 250 colonies per plate andtransferred to nylon membranes (Hybond-N™; Amersham Corporation,Arlington Heights, Ill.). Five filters were lifted for a total of ¹⁸1250 colonies. The filters were marked with a hot needle fororientation, then denatured for 6 minutes in 0.5 M NaOH and 1.5 MTris-HCl, pH 7.2. The filters were then neutralized in 1.5 M NaCl and0.5 M Tris-HCl, pH 7.2 for 6 minutes. The DNA was fixed to the filtersusing a a UV crosslinker (Stratalinker®, Stratagene, La Jolla, Calif.)at 1200 joules. A probe was generated by PCR using oligonucleotideprimers zc21,223 (SEQ ID NO:40) and zc21,224 (SEQ ID NO:39), and a mouse15-day embryo template at an annealing temperature of 66° C. for 35cycles. The PCR fragment was gel purified using a spin column containinga silica gel membrane (QIAquick™ Gel Extraction Kit; Qiagen, Inc.,Valencia, Calif.). The DNA was radioactively labeled with ³²P using acommercially available kit (Rediprime™ II random-prime labeling system;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a commercially availablepush column (NucTrap® column; Stratagene, La Jolla, Calif.; see U.S.Pat. No. 5,336,412). The filters were prewashed at 65° C. in prewashbuffer consisting of 0.25×SSC, 0.25% SDS and 1 mM EDTA. The solution waschanged a total of three times over a 45-minute period to remove celldebris. Filters were prehybridized overnight at 65° C. in 25 ml of ahybridization solution (ExpressHyb™ Hybridization Solution; ClontechLaboratories, Inc., Palo Alto, Calif.), then hybridized overnight at 65°C. in the same solution. Filters were rinsed twice at 65° C. in pre-washbuffer (0.25×SSC, 0.25% SDS, and 1 mM EDTA), then washed twice inpre-wash buffer at 65° C. Filters were exposed to film for 2 days at−80° C. There were 10 positives on the filters. 3 clones were pickedfrom the positive areas, streaked out, and 15 individual colonies fromthese-three positives were screened by PCR using primers zc21,223 (SEQID NO:40) and zc21,334 (SEQ ID NO:41) at an annealing temp of 66° C. Twopositives were recovered and sequenced. Both sequences were found to bethe same and encoded full-length mouse zvegf3 (SEQ ID NO:42).

The amino acid sequence is highly conserved between mouse and humanzvegf3s, with an overall amino acid sequence identity of 87%. Thesecretory peptide, CUB domain, inter-domain, and growth factor domainhave 82%, 92%, 79% and 94% amino acid identity, respectively.

Example 5

Northern blotting was performed using Mouse Multiple Tissue Blots fromOrigene, Rockville, Md. and Clontech, Palo Alto, Calif. An approximately800-bp DNA probe was generated by PCR using primer zc21,223 (SEQ IDNO:40) for the 5′ end and primer zc21,224 (SEQ ID NO:39) for the 3′ end.The reaction was run for 35 cycles at an annealing temperature of 66° C.using Ex Taq™ DNA polymerase (PanVera). The reaction product was gelpurified using a spin column containing a silica gel membrane andlabeled with ³²P using a commercially available labeling kit(Multiprime™ DNA labeling system, Amersham Corp.) according to themanufacturer's specifications. The labeled probe was purified using apush column. A commercially available hybridization solution(ExpressHyb™ Hybridization Solution; Clontech Laboratories, Inc.) wasused for prehybridization and as a hybridizing solution for the Northernblots. Hybridization took place overnight at 65° C., then the blots werewashed 4 times in 2×SCC and 0.05 SDS at room temperature, followed bytwo washes in 0.1×SSC and 0.1% SDS at 50° C. The blots were then exposedto film two days and overnight at −80°C. Multiple transcript sizes wereobserved. Transcripts of ^(˜)3.5 and ^(˜)4.0 kb were seen in 7, 11, 15and 17-day embryo, with the strongest strongest at 7 days and taperingoff at 17 days. A ^(˜)3.0 kb transcript was seen in kidney, liver,brain, and possibly testis. A ^(˜)1.0 kb transcript was seen in testis,muscle, and spleen. Dot blots corresponded to the northern blots.

Example 6

To make transgenic animals expressing zvegf3 genes requires adult,fertile males (studs) (B6C3f1, 2-8 months of age (Taconic Farms,Germantown, N.Y.)), vasectomized males (duds) (B6D2f1, 2-8 months,(Taconic Farms)), prepubescent fertile females (donors) (B6C3f1, 4-5weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2f1,2-4 months, (Taconic Farms)).

The donors are acclimated for 1 week, then injected with approximately 8IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma, St. Louis, Mo.)I.P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin(hCG (Sigma)) I.P. to induce superovulation. Donors are mated with studssubsequent to hormone injections. Ovulation generally occurs within 13hours of hCG injection. Copulation is confirmed by the presence of avaginal plug the morning following mating.

Fertilized eggs are collected under a surgical scope (Leica MZ12 StereoMicroscope, Leica, Wetzlar, Germany). The oviducts are collected andeggs are released into urinanalysis slides containing hyaluronidase(Sigma Chemical Co.). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (Table 7; all reagents available from SigmaChemical Co.) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are stored in a 37° C./5% CO₂ incubator untilmicroinjection.

TABLE 7 mgs/200 ml mgs/500 ml NaCl 1280 3200 KCl 72 180 KH₂PO₄ 32 80MgSO₄ · 7H₂O 60 150 Glucose 200 500 Ca²⁺ Lactate 106 265Benzylpenicillin 15 37.5 Streptomycin SO₄ 10 25 NaHCO₃ 380 950 NaPyruvate 5 12.5 H₂O 200 ml 500 ml 500 mM EDTA 100 μl 250 μl 5% PhenolRed 200 μl 500 μl BSA 600 1500

Zvegf3 cDNA is inserted into the expression vector pHB12-8 (see FIG. 2).Vector pHB12-8 was derived from p2999B4 (Palmiter et al., Mol. CellBiol. 13:5266-5275, 1993) by insertion of a rat insulin II intron (ca.200 bp) and polylinker (Fse I/Pme I/Asc I) into the Nru I site. Thevector comprises a mouse metallothionein (MT-1) promoter (ca. 750 bp)and human growth hormone (hGH) untranslated region and polyadenylationsignal (ca. 650 bp) flanked by 10 kb of MT-1 5′ flanking sequence and 7kb of MT-1 3′ flanking sequence. The cDNA is inserted between theinsulin II and hGH sequences.

10-20 micrograms of plasmid DNA is linearized, gel-purified, andresuspended in 10 mM Tris pH 7.4, 0.25 mM EDTA pH 8.0, at a finalconcentration of 5-10 nanograms per microliter for microinjection.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary) and injected into individual eggs. Eachegg is penetrated with the injection needle into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle is withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pregassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, 2-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy 2-cell embryos from theprevious day's injection are transferred into the recipient. The swollenampulla is located and holding the oviduct between the ampulla and thebursa, and a nick in the oviduct is made with a 28 g needle close to thebursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans are allowed to slide in. The peritoneal wall is closed with onesuture, and the skin is closed with a wound clip. The mice recuperate ona 37° C. slide warmer for a minimum of 4 hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using a commerciallyavailable kit (DNeaSy™ 96 Tissue Kit; Qiagen, Valencia, Calif.)following the manufacturer's instructions. Genomic DNA is analyzed byPCR using primers designed to the human growth hormone (hGH) 3′ UTRportion of the transgenic vector. The use of a region unique to thehuman sequence (identified from an alignment of the human and mousegrowth hormone 3′ UTR DNA sequences) ensures that the PCR reaction doesnot amplify the mouse sequence. Primers zc17,251 (SEQ ID NO:30) andzc17,252 (SEQ ID NO:31) amplify a 368-base-pair fragment of hGH. Inaddition, primers zc17,156 (SEQ ID NO:32) and zc17,157 (SEQ ID NO:33),which hybridize to vector sequences and amplify the cDNA insert, may beused along with the hGH primers. In these experiments, DNA from animalspositive for the transgene will generate two bands, a 368-base-pair bandcorresponding to the hGH 3′ UTR fragment and a band of variable sizecorresponding to the cDNA insert.

Once animals are confirmed to be transgenic (TG), they are back-crossedinto an inbred strain by placing a TG female with a wild-type male, or aTG male with one or two wild-type female(s). As pups are born andweaned, the sexes are separated, and their tails snipped for genotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the xiphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum, and the left lateral lobeof the liver is exteriorized. Using 4-0 silk, a tie is made around thelower lobe securing it outside the body cavity. An atraumatic clamp isused to hold the tie while a second loop of absorbable Dexon (AmericanCyanamid, Wayne, N.J.) is placed proximal to the first tie. A distal cutis made from the Dexon tie, and approximately 100 mg of the excisedliver tissue is placed in a sterile petri dish. The excised liversection is transferred to a 14-ml polypropylene round bottom tube, snapfrozen in liquid nitrogen, and stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage is placed on a37° C. heating pad for 24 hours post-operatively. The animal is checkeddaily post-operatively, and the wound clips are removed 7-10 days aftersurgery.

Analysis of the mRNA expression level of each transgene is done using anRNA solution hybridization assay or real-time PCR on an ABI Prism 7700(PE Applied Biosystems, Inc., Foster City, Calif.) following themanufacturer's instructions.

An adenovirus vector was prepared using a liver-specific albumin geneenhancer and basal promoter (designated “AEO promoter”). The albuminpromoter construct (designated pAEO) was constructed by inserting a 2.2kb Not1/EcoRV fragment from pALBdelta2L (Pinkert et al., Genes Dev.1:268-276, 1987) and an 850 bp NruI/Not1 DNA segment comprising the ratinsulin II intron, an FseI/PmeI/AscI polylinker, and the human growthhormone poly A sequence into a commercially available phagemid vector(pBluescript® KS(+); Stratagene, La Jolla, Calif.). For microinjection,the plasmid is digested with Notl to liberate the expression cassette.

An additional adenovirus vector was constructed using an epithelialcell-specific keratin gene (K14) promoter (Vassar et al., Proc. Natl.Acad. Sci. USA 86:1563-1567, 1989). The 1038-bp open reading frameencoding full-length human zvegf3 was amplified by PCR so as tointroduce an optimized initiation codon and flanking 5′ PmeI and 3′ AscIsites using the primers ZC20,180 (SEQ ID NO:34) and ZC20,181 (SEQ IDNO:35). The resulting PmeI/AscI fragment was subcloned into thepolylinker of pKFO114, a basal keratinocyte-restricted transgenic vectorcomprising the human keratin 14 (K14) promoter (an approximately 2.3 Kbfragment amplified from human genomic DNA [obtained from ClontechLaboratories, Inc.] based on the sequence of Staggers et al., “Sequenceof the promoter for the epidermal keratin gene, K14”, GenBank accession#U11076, 1994), followed by a heterologous intron (a 294-bp BstXI/PstIfragment from pIRES1hyg (Clontech Laboratories, Inc.; see, Huang andGorman, Nucleic Acids Res. 18:937-947, 1990), a PmeI/AscI polylinker,and the human growth hormone gene polyadenylation signal (a 627 bpSmaI/EcoRI fragment; see, Seeburg, DNA 1:239-249, 1982). The transgeneinsert was separated from the plasmid backbone by NotI digestion andagarose gel purification, and fertilized ova from matings of B6C3F1Tacmice or inbred FVB/NTac mice were microinjected and implanted intopseudopregnant females essentially as described by Malik et al., Molec.Cell. Biol. 15:2349-2358, 1995. Transgenic founders were identified byPCR on genomic tail DNA using primers specific for the human growthhormone poly A signal (ZC17,252, SEQ ID NO:31; and ZC17,251, SEQ IDNO:30) to amplify a 368-bp diagnostic product. Transgenic lines wereinitiated by breeding founders with C57BL/6Tac or FVB/NTac mice.

Transgenic mice were generated essentially as disclosed above usingMT-1, K14, and AEO promoters. Four MT-1/zvegf3 transgenic mice weregenerated. In one animal (female) approximately 800 molecules zvegf3mRNA/cell were produced in the liver after zinc induction. This animalhad enlargement of the liver and spleen. Also observed wereproliferation of hepatic sinusoidal cells and extra-medullaryhematopoiesis. One K14/zvegf3 transgenic mouse (female) showed a lowlevel of expression with low body weight, low hematocrit, and lowplatelet count. One AEO/zvegf3 transgenic mouse (male) with a low levelof expression exhibited liver sinusoidal cell proliferation.

Example 7

An expression plasmid containing all or part of a polynucleotideencoding zvegf3 is constructed via homologous recombination. A fragmentof zvegf3 cDNA is isolated by PCR using the polynucleotide sequence ofSEQ ID NO:1 with flanking regions at the 5′ and 3′ ends corresponding tothe vector sequences flanking the zvegf3 insertion point. The primersfor PCR each include from 5′ to 3′ end: 40 bp of flanking sequence fromthe vector and 17 bp corresponding to the amino and carboxyl terminifrom the open reading frame of zvegf3.

Ten μl of the 100 μl PCR reaction is run on a 0.8%low-melting-temperature agarose (SeaPlaque GTG®; FMC BioProducts,Rockland, Me.) gel with 1×TBE buffer for analysis. The remaining 90 μlof PCR reaction is precipitated with the addition of 5 μl 1 M NaCl and250 μl of absolute ethanol. The plasmid pZMP6, which has been cut withSmaI, is used for recombination with the PCR fragment. Plasmid pZMP6 wasconstructed from pZP9 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, underAccession No. 98668) with the yeast genetic elements taken from pRS316(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession No. 77145), aninternal ribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain. pZMP6 is a mammalian expression vector containingan expression cassette having the cytomegalovirus immediate earlypromoter, multiple restriction sites for insertion of coding sequences,a stop codon, and a human growth hormone terminator. The plasmid alsocontains an E. coli origin of replication; a mammalian selectable markerexpression unit comprising an SV40 promoter, enhancer and origin ofreplication, a DHFR gene, and the SV40 terminator; as well as the URA3and CEN-ARS sequences required for selection and replication in S.cerevisiae.

One hundred microliters of competent yeast (S. cerevisiae) cells areindependently combined with 10 μl of the various DNA mixtures from aboveand transferred to a 0.2-cm electroporation cuvette. The yeast/DNAmixtures are electropulsed using power supply settings of 0.75 kV (5kV/cm), ∞ ohms, 25 μF. To each cuvette is added 600 μl of 1.2 Msorbitol, and the yeast is plated in two 300-μl aliquots onto two URA-Dplates and incubated at 30° C. After about 48 hours, the Ura⁺ yeasttransformants from a single plate are resuspended in 1 ml H₂O and spunbriefly to pellet the yeast cells. The cell pellet is resuspended in 1ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture is addedto an Eppendorf tube containing 300 μl acid-washed glass beads and 200μl phenol-chloroform, vortexed for 1 minute intervals two or threetimes, and spun for 5 minutes in an Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase is transferred toa fresh tube, and the DNA is precipitated with 600 μl ethanol (EtOH),followed by centrifugation for 10 minutes at 4° C. The DNA pellet isresuspended in 10 μl H₂O.

Transformation of electrocompetent E. coli host cells (Electromax DH10B™cells; obtained from Life Technologies, Inc., Gaithersburg, Md.) is donewith 0.5-2 ml yeast DNA prep and 40 ul of cells. The cells areelectropulsed at 1.7 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) is plated in 250-μl aliquots on four LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual clones harboring the correct expression construct for zvegf3are identified by restriction digest to verify the presence of thezvegf3 insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The inserts of positive clones aresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing a commercially available kit (QIAGEN Plasmid Maxi Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstruct is designated pZMP6/zvegf3 (FIG. 3).

Example 8

CHO DG44 cells (Chasin et al., Som. Cell. Molec. Genet. 12:555-666,1986) are plated in 10-cm tissue culture dishes and allowed to grow toapproximately 50% to 70% confluency overnight at 37° C., 5% CO₂, inHam's F12/FBS media (Ham's F12 medium, Life Technologies), 5% fetalbovine serum (Hyclone, Logan, Utah), 1% L-glutamine (JRH Biosciences,Lenexa, Kans.), 1% sodium pyruvate (Life Technologies). The cells arethen transfected with the plasmid pZMP6/zvegf3 by liposome-mediatedtransfection using a 3:1 (w/w) liposome formulation of the polycationiclipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium-trifluoroacetateand the neutral lipid dioleoyl phosphatidylethanolamine inmembrane-filetered water (Lipofectamine™ Reagent, Life Technologies), inserum free (SF) media formulation (Ham's F12, 10 mg/ml transferrin, 5mg/ml insulin, 2 mg/ml fetuin, 1% L-glutamine and 1% sodium pyruvate).Plasmid pZMP6/Zvegf3 is diluted into 15-ml tubes to a total final volumeof 640 μl with SF media. 35 μl of Lipofectamine™ is mixed with 605 μl ofSF medium. The Lipofectamine™ mixture is added to the DNA mixture andallowed to incubate approximately 30 minutes at room temperature. Fiveml of SF media is added to the DNA:Lipofectamine™ mixture. The cells arerinsed once with 5 ml of SF media, aspirated, and the DNA:Lipofectamine™mixture is added. The cells are incubated at 37° C. for five hours, then6.4 ml of Ham's F12/10% FBS, 1% PSN media is added to each plate. Theplates are incubated at 37° C. overnight, and the DNA:Lipofectamine™mixture is replaced with fresh 5% FBS/Ham's media the next day. On day 3post-transfection, the cells are split into T-175 flasks in growthmedium. On day 7 postransfection, the cells are stained withFITC-anti-CD8 monoclonal antibody (Pharmingen, San Diego, Calif.)followed by anti-FITC-conjugated magnetic beads (Miltenyi Biotec). TheCD8-positive cells are separated using commercially available columns(mini-MACS columns; Miltenyi Biotec) according to the manufacturer'sdirections and put into DMEM/Ham's F12/5% FBS without nucleosides butwith 50 nM methotrexate (selection medium).

Cells are plated for subcloning at a density of 0.5, 1 and 5 cells perwell in 96-well dishes in selection medium and allowed to grow out forapproximately two weeks. The wells are checked for evaporation of mediumand brought back to 200 μl per well as necessary during this process.When a large percentage of the colonies in the plate are nearconfluency, 100 μl of medium is collected from each well for analysis bydot blot, and the cells are fed with fresh selection medium. Thesupernatant is applied to a nitrocellulose filter in a dot blotapparatus, and the filter is treated at 100° C. in a vacuum oven todenature the protein. The filter is incubated in 625 mM Tris-glycine, pH9.1, 5 mM β-mercaptoethanol, at 65° C., 10 minutes, then in 2.5% non-fatdry milk Western A Buffer (0.25% gelatin, 50 mM Tris-HCl pH 7.4, 150 mMNaCl, 5 mM EDTA, 0.05% Igepal CA-630) overnight at 4° C. on a rotatingshaker. The filter is incubated with the antibody-HRP conjugate in 2.5%non-fat dry milk Western A buffer for 1 hour at room temperature on arotating shaker. The filter is then washed three times at roomtemperature in PBS plus 0.01% Tween 20, 15 minutes per wash. The filteris developed with chemiluminescence reagents (ECL™ direct labelling kit;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sdirections and exposed to film (Hyperfilm ECL, Amersham) forapproximately 5 minutes. Positive clones are trypsinized from the96-well dish and transferred to 6-well dishes in selection medium forscaleup and analysis by Western blot.

Example 9

Full-length zvegf3 protein was produced in BHK cells transfected withpZMP6/zvegf3 (Example 7). BHK 570 cells (ATCC CRL-10314) were plated in10-cm tissue culture dishes and allowed to grow to approximately 50 to70% confluence overnight at 37° C., 5% CO₂, in DMEM/FBS media (DMEM,Gibco/BRL High Glucose; Life Technologies), 5% fetal bovine serum(Hyclone, Logan, Utah), 1 mM L-glutamine (JRH Biosciences, Lenexa,Kans.), 1 mM sodium pyruvate (Life Technologies). The cells were thentransfected with pZMP6/zvegf3 by liposome-mediated transfection (using(Lipofectamine™; Life Technologies), in serum free (SF) media (DMEMsupplemented with 10 mg/ml transferrin, 5 mg/ml insulin, 2 mg/ml fetuin,1% L-glutamine and 1% sodium pyruvate). The plasmid was diluted into15-ml tubes to a total final volume of 640 μl with SF media. 35 μl ofthe lipid mixture was mixed with 605 μl of SF medium, and the mixturewas allowed to incubate approximately 30 minutes at room temperature.Five milliliters of SF media was added to the DNA:lipid mixture. Thecells were rinsed once with 5 ml of SF media, aspirated, and theDNA:lipid mixture was added. The cells were incubated at 37° C. for fivehours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to eachplate. The plates were incubated at 37° C. overnight, and the DNA:lipidmixture was replaced with fresh 5% FBS/DMEM media the next day. On day 5post-transfection, the cells were split into T-162 flasks in selectionmedium (DMEM+5% FBS, 1% L-Gln, 1% NaPyr, 1 μM methotrexate).Approximately 10 days post-transfection, two 150-mm culture dishes ofmethotrexate-resistant colonies from each transfection were trypsinized,and the cells are pooled and plated into a T-162 flask and transferredto large-scale culture.

Example 10

A mammalian cell expression vector for the growth factor domain ofzvegf3 was constructed essentially as disclosed in Example 7. The codingsequence for the growth factor domain (residues 235-345 of SEQ ID NO:2),joined to a sequence encoding an optimized t-PA secretory signalsequence (U.S. Pat. No. 5,641,655) was joined to the linearized pZMP11vector downstream of the CMV promoter. The plasmid pZMP11 is a mammalianexpression vector containing an expression cassette having the CMVimmediate early promoter, a consensus intron from the variable region ofmouse immunoglobulin heavy chain locus, Kozak sequences, multiplerestriction sites for insertion of coding sequences, a stop codon, and ahuman growth hormone terminator. The plasmid also contains an IRESelement from poliovirus, the extracellular domain of CD8 truncated atthe C-terminal end of the transmembrane domain, an E. coli origin ofreplication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a DHFR gene, the SV40terminator, and the URA3 and CEN-ARS sequences required for selectionand replication in S. cerevisiae. The resulting vector, designatedpZMP11/zv3GF-otPA, is shown in FIG. 4.

BHK 570 cells were transfected with pZMP11/zv3GF-otPA and culturedessentially as disclosed in Example 9.

Example 11

For construction of adenovirus vectors, the protein coding region ofhuman zvegf3 was amplified by PCR using primers that added PmeI and AscIrestriction sties at the 5′ and 3′ termini respectively. PCR primersZC20,180 (SEQ ID NO:34) and ZC20,181 (SEQ ID NO:35) were used with afull-length zvegf3 cDNA template in a PCR reaction as follows: one cycleat 95° C. for 5 minutes; followed by 15 cycles at 95° C. for 1 min., 61°C. for 1 min., and 72° C. for 1.5 min.; followed by 72° C. for 7 min.;followed by a 4° C. soak. The PCR reaction product was loaded onto a1.2% low-melting-temperature agarose gel in TAE buffer (0.04 MTris-acetate, 0.001 M EDTA). The zvegf3 PCR product was excised from thegel and purified using a commercially available kit comprising a silicagel mambrane spin column (QIAquick™ PCR Purification Kit and gel cleanupkit; Qiagen, Inc.) as per kit instructions. The PCR product was thendigested with PmeI and AscI, phenol/chloroform extracted, EtOHprecipitated, and rehydrated in 20 ml TE (Tris/EDTA pH 8). The 1038 bpzvegf3 fragment was then ligated into the PmeI-AscI sites of thetransgenic vector pTG12-8 (also known as pHB12-8; see Example 6) andtransformed into E. coli DH10B™ competent cells by electroporation.Clones containing zvegf3 were identified by plasmid DNA miniprepfollowed by digestion with PmeI and AscI. A positive clone was sequencedto insure that there were no deletions or other anomalies in theconstruct. The sequence of zvegf3 cDNA was confirmed.

DNA was prepared using a commercially available kit (Maxi Kit, Qiagen,Inc.), and the 1038bp zvegf3 cDNA was released from the pTG12-8 vectorusing PmeI and AscI enzymes. The cDNA was isolated on a 1% low meltingtemperature agarose gel and was excised from the gel. The gel slice wasmelted at 70° C., and the DNA was extracted twice with an equal volumeof Tris-buffered phenol and precipitated with EtOH. The DNA wasresuspended in 10 μl H₂O.

The zvegf3 cDNA was cloned into the EcoRV-AscI sites of a modifiedpAdTrack-CMV (He, T -C. et al., Proc. Natl. Acad. Sci. USA 95:2509-2514,1998). This construct contains the green fluorescent protein (GFP)marker gene. The CMV promoter driving GFP expression was replaced withthe SV40 promoter, and the SV40 polyadenylation signal was replaced withthe human growth hormone polyadenylation signal. In addition, the nativepolylinker was replaced with FseI, EcoRV, and AscI sites. This modifiedform of pAdTrack-CMV was named pZyTrack. Ligation was performed using acommercially available DNA ligation and screening kit (Fast-Link™ kit;Epicentre Technologies, Madison, Wis.). Clones containing zvegf3 wereidentified by digestion of mini prep DNA with FseI and AscI. In order tolinearize the plasmid, approximately 5 μg of the resulting pzyTrackzvegf3 plasmid was digested with PmeI. Approximately 1 μg of thelinearized plasmid was cotransformed with 200 ng of supercoiled pAdEasy(He et al., ibid.) into E. coli BJ5183 cells (He et al., ibid.). Theco-transformation was done using a Bio-Rad Gene Pulser at 2.5 kV, 200ohms and 25 μFa. The entire co-transformation mixture was plated on 4 LBplates containing 25 μg/ml kanamycin. The smallest colonies were pickedand expanded in LB/kanamycin, and recombinant adenovirus DNA wasidentified by standard DNA miniprep procedures. Digestion of therecombinant adenovirus DNA with FseI and AscI confirmed the presence ofthe zvegf3 insert. The recombinant adenovirus miniprep DNA wastransformed into E. coli DH10B™ competent cells, and DNA was preparedusing a Maxi Kit (Qiagen, Inc.) aaccording to kit instructions.

Approximately 5 μg of recombinant adenoviral DNA was digested with PacIenzyme (New England Biolabs) for 3 hours at 37° C. in a reaction volumeof 100 μl containing 20-30U of PacI. The digested DNA was extractedtwice with an equal volume of phenol/chloroform and precipitated withethanol. The DNA pellet was resuspended in 10 μl distilled water. A T25flask of QBI-293A cells (Quantum Biotechnologies, Inc. Montreal, Qc.Canada), inoculated the day before and grown to 60-70% confluence, weretransfected with the PacI digested DNA. The PacI-digested DNA wasdiluted up to a total volume of 50 μl with sterile HBS (150 mM NaCl, 20mM HEPES). In a separate tube, 20 μl of 1 mg/mlN-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium salts (DOTAP)(Boehringer Mannheim, Indianapolis, Ind.) was diluted to a total volumeof 100 μl with HBS. The DNA was added to the DOTAP, mixed gently bypipeting up and down, and left at room temperature for 15 minutes. Themedia was removed from the 293A cells and washed with 5 ml serum-freeminimum essential medium (MEM) alpha containing 1 mM sodium pyruvate,0.1 mM MEM non-essential amino acids, and 25 mM HEPES buffer (reagentsobtained from Life Technologies, Gaithersburg, Md.). 5 ml of serum-freeMEM was added to the 293A cells and held at 37° C. The DNA/lipid mixturewas added drop-wise to the T25 flask of 293A cells, mixed gently, andincubated at 37° C. for 4 hours. After 4 h the media containing theDNA/lipid mixture was aspirated off and replaced with 5 ml complete MEMcontaining 5% fetal bovine serum. The transfected cells were monitoredfor GFP expression and formation of foci (viral plaques).

Seven days after transfection of 293A cells with the recombinantadenoviral DNA, the cells expressed the GFP protein and started to formfoci (viral “plaques”). The crude viral lysate was collected using acell scraper to collect all of the 293A cells. The lysate wastransferred to a 50-ml conical tube. To release most of the virusparticles from the cells, three freeze/thaw cycles were done in a dryice/ethanol bath and a 37° waterbath.

The crude lysate was amplified (Primary (1°) amplification) to obtain aworking “stock” of zvegf3 rAdV lysate. Ten 10-cm plates of nearlyconfluent (80-90%) 293A cells were set up 20 hours previously, 200 μl ofcrude rAdV lysate added to each 10-cm plate and monitored for 48 to 72hours looking for CPE under the white light microscope and expression ofGFP under the fluorescent microscope. When all of the 293A cells showedCPE (Cytopathic Effect) this 1° stock lysate was collected andfreeze/thaw cycles performed as described under Crude rAdV Lysate.

Secondary (2°) amplification of zvegf3 rAdV was obtained as follows:Twenty 15-cm tissue culture dishes of 293A cells were prepared so thatthe cells were 80-90% confluent. All but 20 ml of 5% MEM media wasremoved, and each dish was inoculated with 300-500 μl of the 1°amplified rAdv lysate. After 48 hours the 293A cells were lysed fromvirus production, the lysate was collected into 250-ml polypropylenecentrifuge bottles, and the rAdV was purified.

NP-40 detergent was added to a final concentration of 0.5% to thebottles of crude lysate in order to lyse all cells. Bottles were placedon a rotating platform for 10 minutes agitating as fast as possiblewithout the bottles falling over. The debris was pelleted bycentrifugation at 20,000×G for 15 minutes. The supernatant wastransferred to 250-ml polycarbonate centrifuge bottles, and 0.5 volumeof 20% PEG8000/2.5 M NaCl solution was added. The bottles were shakenovernight on ice. The bottles were centrifuged at 20,000×G for 15minutes and, the supernatant was discarded into a bleach solution. Usinga sterile cell scraper, the white, virus/PEG precipitate from 2 bottleswas resuspended in 2.5 ml PBS. The resulting virus solution was placedin 2-ml microcentrifuge tubes and centrifuged at 14,000×G in themicrocentrifuge for 10 minutes to remove any additional cell debris. Thesupernatant from the 2-ml microcentrifuge tubes was transferred into a15-ml polypropylene snapcap tube and adjusted to a density of 1.34 g/mlwith CsCl. The volume of the virus solution was estimated, and 0.55 g/mlof CsCl was added. The CsCl was dissolved, and 1 ml of this solutionweighed 1.34 g. The solution was transferred to 3.2-ml, polycarbonate,thick-walled centrifuge tubes and spun at 348,000×G for 3-4 hours at 25°C. The virus formed a white band. Using wide-bore pipette tips, thevirus band was collected.

The virus from the gradient had a large amount of CsCl which had to beremoved before it was used on cells. Commercially available ion-exchangecolumns (PD-10 columns prepacked with Sephadex® G-25M; PharmaciaBiotech, Piscataway, N.J.) were used to desalt the virus preparation.The column was equilibrated with 20 ml of PBS. The virus was loaded andallowed to run into the column. 5 ml of PBS was added to the column, andfractions of 8-10 drops were collected. The optical densities of 1:50dilutions of each fraction were determined at 260 nm on aspectrophotometer. A clear absorbance peak was present between fractions7-12. These fractions were pooled, and the optical density (OD) of a1:25 dilution was determined. OD was converted to virus concentrationusing the formula: (OD at 260 nm) (25) (1.1×10¹²)=virions/ml. The OD ofa 1:25 dilution of the zvegf3 rAdV was 0.145, giving a virusconcentration of 4×10¹² virions/ml.

To store the virus, glycerol was added to the purified virus to a finalconcentration of 15%, mixed gently but effectively, and stored inaliquots at −80° C.

A protocol developed by Quantum Biotechnologies, Inc. (Montreal, Canada)was followed to measure recombinant virus infectivity. Briefly, two96-well tissue culture plates were seeded with 1×10⁴ 293A cells per wellin MEM containing 2% fetal bovine serum for each recombinant virus to beassayed. After 24 hours 10-fold dilutions of each virus from 1×10⁻² to1×10⁻¹⁴ were made in MEM containing 2% fetal bovine serum. 100 μl ofeach dilution was placed in each of 20 wells. After 5 days at 37° C.,wells were read either positive or negative for Cytopathic Effect (CPE)and a value for “Plaque Forming Units/ml” (PFU) was calculated.

TCID₅₀ formulation used was as per Quantum Biotechnologies, Inc., above.The titer (T) was determined from a plate where virus was diluted from10⁻² to 10⁻¹⁴, and read 5 days after the infection. At each dilution aratio (R) of positive wells for CPE per the total number of wells wasdetermined.

To calculate titer of the undiluted virus sample: the factor,“F”=1+d(S−0.5); where “S” is the sum of the ratios (R); and “d” isLog10of the dilution series, for example, “d” is equal to 1 for aten-fold dilution series. The titer of the undiluted sample isT=10^((1+F))=TCID₅₀/ml. To convert TCID₅₀/ml to pfu/ml, 0.7 issubtracted from the exponent in the calculation for titer (T).

The zvegf3 adenovirus had a titer of 1.8×10¹⁰ pfu/ml.

Example 12

Treatment of mice with zvegf3-adenovirus led to changes in liver andspleen. The livers were pale and very enlarged, with enlarged vessels atthe tips of the lobes. The livers also showed sinusoidal cellproliferation. Changes were also seen in hepatocytes (hypertrophy,degeneration, and necrosis) and were most likely non-specific effects ofadenovirus infection. Splenic change consisted of increasedextramedulary hematopoiesis, which was correlated with enlarged splenicsize.

Example 13

Data from adenovirus-treated and transgenic mice were consistent withincreased hematopoiesis and/or angiogenesis in the test animals. A studywas therefore undertaken to test whether adenovirally delivered zvegf3stimulated cells in the hematopoietic or lymphoid compartments toproliferate, as determined by examination of peripheral blood,histological examination, and incorporation of bromodeoxy uridine (BrdU)into tissues.

Mice (male, C57Bl, 7 weeks old) were divided into three groups. On day0, parental or zvegf3 adenovirus was administered to the first (n=11)and second (n=12) groups, respectively, via the tail vein, with eachmouse receiving a dose of ^(˜)1×10¹¹ particles in ^(˜)0.1 ml volume. Thethird group (n=8) received no treatment. Each mouse was given twointraperitoneal doses of 3 mg of freshly made BrdU solution atapproximately 24 and 12 hours prior to sacrifice. On days 2, 4, 6, 8,and 10, two mice from each treatment group and one or two untreated micewere sacrificed, and tissues and blood were harvested. Samples wereanalyzed for complete blood count (CBC) and serum chemistry, and slideswere prepared for manual differential blood and marrow progenitor cellanalysis. One femur, lung, heart, thymus, liver, kidney, spleen,pancreas, duodenum, and mesenteric lymph nodes were submitted forstandard histology and assessment of BrdU incorporation. The lining ofthe duodenum served as the control tissue for BrdU incorporation.

In addition, two mice that received approximately half the dose ofzvegf3 adenovirus particles and one mouse that received the full dose ofparental adenovirus were sacrificed and analyzed as described above onday 16.

A piece of liver from each mouse was saved for mRNA assay of adenovirusprotein to examine the time course of expression of the adenoviruspreparations.

Beginning on day 6, most of the animals treated with either adenovirushad visibly enlarged livers and spleens compared to the untreated mice.The livers of the zvegf3 adenovirus-treated mice tended to look morepale than animals treated with the parental virus. Proliferation ofsinusoidal cells was observed in liver. Visual inspection suggested thatthese cells were stellate cells and/or fibroblasts. Spleen color was thesame in both groups. Most of the animals that received the zvegf3adenovirus had paler femur shafts, with the marrow lighter in color.

Peripheral blood CBCs showed a possible difference in platelet counts,but not in RBC or WBC counts between zvegf3 and parental virus-treatedanimals. In comparison to the untreated and partental virus-treatedgroups, the zvegf3 group had lower platelet counts on days 2, 4, 6, and8, but not on day 10. The mean platelet volume (average size ofindividual platelets) in the zvegf3 group also tended to be greater,consistent with a relative increase in the larger, immature plateletpopulation.

BrdU labeling showed increased cell proliferation in kidney, mainlyin-the medulla and to a lesser extent in the cortex. Proliferating cellsappeared to be interstitial cells, which may have included fibroblastsand/or mesangial cells.

Example 14

Zvegf3 was assayed in an aortic ring outgrowth assay (Nicosia andOttinetti, Laboratory Investigation 63(1):115, 1990; Villaschi andNicosia, Am. J. Pathology 143(1):181-190, 1993). Thoracic aortas wereisolated from 1-2 month old SD male rats and transferred to petri dishescontaining HANK's buffered salt solution. The aortas were flushed withadditional HANK's buffered salt solution to remove blood, andadventitial tissue surrounding the aorta was carefully removed. Cleanedaortas were transferred to petri dishes containing EBM basal media,serum free (Clonetics, San Diego, Calif.). Aortic rings were obtained byslicing approximately 1-mm sections using a scalpel blade. The ends ofthe aortas used to hold the aorta in place were not used. The rings wererinsed in fresh EBM basal media and placed individually in a wells of a24-well plate coated with Matrigel (Becton Dickinson, Bedford, Mass.).The rings were overlayed with an additional 50 μl Matrigel and placed at37° C. for 30 minutes to allow the matrix to gel. Test samples werediluted in EBM basal serum-free media supplemented with 100 units/mlpenicillin, 100 μg/ml streptomycin and HEPES buffer and added at 1ml/well. Background control was EBM basal serum-free media alone. BasicFGF (R&D Systems, Minneapolis, Minn.) at 20 ng/ml was used as a positivecontrol. Zvegf33 pZyTrack andenovirus (Example 11) was added to wells,assuming a cell count of 500,000 cells and a multiplicity of infectionof 5000 particles/cell. A null ZyTrack adenovirus (zpar) was used as acontrol. Samples were added in a minimum of quadruplets. Rings wereincubated for 5-7 days at 37° C. and analyzed for growth. Aorticoutgrowth was scored by multiple, blinded observers using 0 as no growthand 4 as maximum growth. Zvegf3 adenovirus produced a significantincrease in outgrowth as compared to controls, and was comparable toother potent growth factors (e.g., bFGF). In additional experiments,purified zvegf3 growth factor domain also caused a signicant increase inoutgrowth at concentrations down to approximately 50 ng/ml.

Zvegf3-responsive cells were stained for alpha smooth muscle actin(characteristic of SMCs), von Willebrand factor (characteristic ofendothelial cells), type I collagen (characteristic of fibroblasts), andvimentin (stains all three cell types). The observed staining patternsindicated that the cells were fibroblasts and smooth muscle cells, withthe possible inclusion of pericytes.

Example 15

Thirty mice (male, c57BL6) were each injected subcutaneously with 2.5×1⁵Lewis lung carcinoma cells (obtained from American Type CultureCollection, Mannassas, Va.). Three days after implantation of cells, themice were split into three groups of ten and were injected with eithersaline, zvegf3 adenovirus (1×10¹¹ particles), or control adenovirus(1×10¹¹ particles). Growth of tumors was monitored by dimensionalmeasurement on day 14 and by gross tumor weight at the time of sacrifice(day 21). Lungs, liver and tumor were examined by histological methods.Tumor size was significantly lower in the zvegf3-treated group comparedto the control adenovirus group (p<0.007), but not significantlydifferent from the saline-treated group (p=0.6). The incidence ofmetastasis was low and did not differ among the groups.

Example 16

Myeloproliferative activity of zvegf3 is tested in a mouse model ofmyelosuppression. On day 0, mice (male C57Bl) in the myelosuppressedgroups are administered 450cGy of radiation and 1.2 mg of Carboplatin. Asecond set of mice remain untreated. Between days 0 and 7, purifiedzvegf3, thrombopoietin, or vehicle control are administeredsubcutaneously. On days 7, 6, 10, 15, and 20 the mice are bled underanasthetic by retro-orbital puncture. Blood samples are analyzed forCBCs, and slides are made for manual differential and immature cellanalysis.

When recovery of blood cell lineages is observed, the animals aresacrificed. Bone marrow is harvested for microscopic analysis and bonemarrow progenitor assays. Spleen and liver are harvested forhistological examination to determine extramedullary hematopoiesis.Lung, thymus, heart, testis, and kidney are analyzed for histology.

Example 17

Polyclonal anti-peptide antibodies were prepared by immunizing twofemale New Zealand white rabbits with the peptides huzvegf3-1 (residues80-104 of SEQ ID NO:2), huzvegf3-2 (residues 299-314 of SEQ ID NO:2),huzvegf3-3 (residues 299-326 of SEQ ID NO:2 with an N-terminal cysresidue), or huzvegf3-4 (residues 195-225 of SEQ ID NO:2 with aC-terminal cys residue). The peptides were synthesized using an AppliedBiosystems Model 431A peptide synthesizer (Applied Biosystems, Inc.,Foster City, Calif.) according to the manufacturer's instructions. Thepeptides huzvegf3-1, huzvegf3-3, and huzvegf3-4 were then conjugated tothe carrier protein maleimide-activated keyhole limpet hemocyanin (KLH)through cysteine residues (Pierce Chemical Co., Rockford, Ill.). Thepeptide huzvefg3-2 was conjugated to the carrier protein KLH usinggluteraldehyde. The rabbits were each given an initial intraperitoneal(IP) injection of 200 μg of conjugated peptide in Complete Freund'sAdjuvant (Pierce Chemical Co.) followed by booster IP injections of 100μg conjugated peptide in Incomplete Freund's Adjuvant every three weeks.Seven to ten days after the administration of the third boosterinjection, the animals were bled and the serum was collected. Therabbits were then boosted and bled every three weeks.

The huzvegf3 peptide-specific antibodies were affinity purified from therabbit serum using an CNBr-Sepharose® 4B peptide column (PharmaciaBiotech) that was prepared using 10 mg of the respective peptides pergram CNBr-Sepharose®, followed by dialysis in PBS overnight. Peptidespecific-huzvegf3 antibodies were characterized by an ELISA titer checkusing 1 μg/ml of the appropriate peptide as an antibody target. Thehuzvegf3-1 peptide-specific antibodies have a lower limit of detection(LLD) of 500 pg/ml by ELISA on its appropriate antibody target andrecognize full-length recombinant protein (MBP-fusion; see Example 28)by ELISA. The huzvegf3-2 peptide-specific antibodies had an LLD of 1ng/ml by ELISA. The huzvegf3-3 peptide-specific antibodies had an LLD of50 pg/ml by ELISA and recognized recombinant protein by Western Blotanalysis. The huzvegf3-4 peptide-specific antibodies had an LLD of 50pg/ml by ELISA and recognized recombinant protein by Western Blotanalysis.

Example 18

Recombinant zvegf3 was analyzed by Western blotting using antibodies tothe huzvegf3-1 and huzvegf3-3 peptides. Protein was produced in BHKcells as disclosed above, and in 293 and MVEC (microvascularendothelial) cells transfected with adenovirus vectors according toconventional methods. Samples were electrophoresed and transferred tonitrocellulose (0.2 μm; Bio-Rad Laboratories, Hercules, Calif.) at roomtemperature using a Hoeffer Scientific Instruments (San Francisco,Calif.) model TE22 blotter with stirring according to directionsprovided in the instrument manual. The transfer was run at 500 mA forone hour or 50 mA for 12 hours in a buffer containing 25 mM Tris base,200 mM glycine, and 20% methanol. The filters were then blocked with 10%non-fat dry milk in buffer A (50 mM Tris (pH 7.4), 5 mM EDTA (pH 8.0),0.05% Igepal CA-630, 150 mM NaCl, 0.25% gelatin) for 10 minutes at roomtemperature. The nitrocellulose was quickly rinsed, then primaryantibody was added in buffer A containing 2.5% non-fat dry milk. Theblots were incubated for 1 hour at room temperature or overnight at 4°C. with gentle shaking or rocking. Following the incubation, blots werewashed three times for 10 minutes each in buffer A. Secondary antibody(goat anti-rabbit IgG conjugated to horseradish peroxidase; obtainedfrom Rockland Inc., Gilbertsville, Pa.) diluted 1:4000 in buffer Acontaining 2.5% non-fat dry milk was added, and the blots were incubatedfor one hour at room temperature with gentle shaking or rocking. Theblots were then washed three times, 10 minutes each, in buffer A, thenquickly rinsed in H₂O. The blots were developed using commerciallyavailable chemiluminescent substrate reagents (SuperSignal® ULTRAreagents 1 and 2 mixed 1:1; reagents obtained from Pierce Chemical Co.),and exposed to film (Hyperfilm ECL™; Amersham Pharmacia Biotech,Piscataway, N.J.) for times ranging from 1 second to 5 minutes or asnecessary.

Using the 3-1 antibody, full-length zvegf3 produced in BHK cells showedtwo bands (M_(r)≈46 kDa and 30 kDa) under reducing conditions. Thelarger band was consistent with the size of the full-length zvegf3monomer, and the smaller band with the CUB domain+interdomain region.Under non-reducing conditions, there was a major band at M_(r)≈78 kDa,which appeared to be the dimerized, full-length molecule. Two smaller,minor bands were also observed. Similar results were obtained with the3—3 antibody, but under reducing conditions the smaller band ran atM_(r)≈22 kDa. Sequence analysis of the smaller band showed it to be theisolated growth factor domain.

The recombinant growth factor domain (produced in BHK cells), analyzedusing the 3—3 antibody, ran as a broad band of M_(r)≈18 kDa underreducing conditions. Under non-reducing conditions, the protein ran astwo bands of M_(r)≈16 and 28 kDa, indicating the presence of bothdimeric and monomeric forms of the growth factor domain.

Zvegf3 protein produced in 293 cells grown in serum-free media showedsizes consistent with the predicted full-length protein. Under reducingconditions, the protein ran at M_(r)≈47 kDa, and under non-reducingconditions at M_(r)≈78 kDa when blots were probed with either antibody3-1 or 3—3. Addition of serum to the media resulted in cleavage of theprotein, as seen in the BHK-produced material.

MVEC cells grown in the presence of 1% serum produced zvegf3 proteinthat ran at M_(r)≈23 kDa under reducing conditions, and M_(r)≈28 kDaunder non-reducing conditions using the 3—3 antibody. These resultsindicate cleavage of the protein, with the antibody recognizing themonomeric and dimeric forms of the growth factor domain. When the MVECcells were adapted to serum-free media, full-length protein wasobserved.

Example 19

Recombinant zvegf3 growth factor domain was produced in BHK 570 cellsgrown in cell factories. Three 15-liter cultures were harvested, and themedia were sterile filtered using a 0.2μ filter. Expression levels wereestimated by western blot analysis of media samples concentrated to 20×vs. 5K cut-off and serially diluted by two-fold to 1.25×. Signalintensity was compared to a signal on the same blot from an MBP-zvegf3fusion protein standard (see Example 28) for which the proteinconcentration had been determined by amino acid analysis. Expressionlevels were consistently between 0.25 and 0.35 mg/L of media.

Protein was purified from conditioned media by a combination of cationexchange chromatography and hydrophobic interaction chromatography.Culture medium was diluted with 0.1 M acetic acid, pH 3.0, containing0.3 M NaCl at a ratio of 60%:40%, (medium:acetic acid) to deliver aprocess stream at 14 mS conductivity and pH 4.0. This stream wasdelivered to a strong cation exchange resin (Poros® HS; PerSeptiveBiosystems, Framingham, Mass.) with a bed volume of 50 ml in a 2-cmdiameter column at a flow rate of 20 ml/minute. A 50-ml bed wassufficient to process 45 L of media and capture all of the targetprotein. Bound protein was eluted, following column washing for 10column volumes in 10 mM acetic acid with 0.15 M NaCl at pH=4.0, byforming a linear gradient to 2M NaCl in 10 mM acetic acid, pH 4.0.Ten-ml fractions were captured into tubes containing 2 ml 2.0 M Tris, pH8.0 to neutralize the acidity. Samples from the cation exchange columnwere analyzed by SDS PAGE with silver staining and western blotting forthe presense of zvegf3. The vegf3 growth domain eluted at 0.2-0.5 MNaCl. Protein-containing fractions were pooled. A 25-ml bed ofchromatography medium (Toso Haas Ether chromatography medium) in a 2 cmdiameter column was equilibrated in 1.8 M (NH₄)₂SO₄ in 25 mM Naphosphate buffer at pH 7.4. The pooled protein from the cation exchangestep was adjusted to 1.8 M (NH₄)₂SO₄ in 25 mM Na phosphate, pH 7.0. Thisstream was flowed over the column at 10 ml/minute. Once the loading wascompleted the column was washed for 10 column volumes with theequilibration buffer prior to eluting with a 10 column volume gradientformed between the equilibration buffer and 40 mM boric acid at pH 8.8.The zvegf3 growth factor domain protein eluted fairly early in thegradient between 1.5 and 1.0 M (NH₄)₂SO₄. At this point the protein was40-60% pure with a major contaminent being insulin-like growth factorbinding protein 4 (IGFBP4).

Protein from the HIC (Ether) chromatography step was applied to a C4reverse-phase HPLC column. The zvegf3 growth factor domain proteineluted at 36% acetonitrile. This material still contained approximately20% (mole/mole) IGFB4.

Example 20

Recombinant zvegf3 growth factor domain is purified fromcell-conditioned media by a combination of cation exchangechromatography, hydrophobic interaction chromatography, and nickelaffinity chromatography. Protein is captured on a strong cation exchangemedium and eluted essentially as disclosed in Example 19. The elutedprotein is further purified by hydrophobic interaction chromatography onan ether resin (Poros® ET; PerSeptive Biosystems). The partiallypurified zvegf3 protein is then bound to a nickel chelate resin at pH7.0-8.0 in 25 mM Na phosphate buffer containing 0.25 M NaCl. The boundprotein is eluted with a descending pH gradient down to pH 5.0 or animidazole gradient. The eluate from the nickel column is adjusted to 1 M(NH₄)₂SO₄, 20 mM MES (morphilino ethanesulfonic acid) at pH 6.0 andpassed through a phenyl ether hydrophobic interaction chromatographycolumn (Poros® PE, PerSeptive Biosystems) that has been equilibrated in1 M (NH₄)₂SO₄, 20 mM MES, pH 6.0. IGFBP4 and minor contaminants areretained on the column. The pass-through fraction, which contains highlypurified zvegf3, is collected. The collected protein is desaltedaccording to conventional methods (e.g., dialysis, ion-exchangechromatography).

Example 21

Recombinant zvegf3 was analyzed for mitogenic activity on human aorticsmooth muscle cells (HAoSMC; Clonetics Corp., Walkersville, Md.) andhuman umbilical vein endothelial cells (HUVEC; Clonetics Corp.). HAoSMCand HUVEC were plated at a density of 5,000 cells/well in 96-wellculture plates and grown for approximately 24 hours in DMEM containing10 % fetal calf serum at 37° C. Cells were quiesced by incubating themfor 24 hours in serum-free DMEM/Ham's F-12 medium containing insulin (5μg/ml), transferrin (20 μg/ml), and selenium (16 pg/ml) (ITS). At thetime of the assay, the medium was removed, and test samples were addedto the wells in triplicate. Test samples consisted of either conditionedmedia (CM) from adenovirally-infected HaCaT human keratinocyte cells(Boukamp et al., J. Cell. Biol. 106:761-771, 1988) expressingfull-length zvegf3, purified growth factor domain expressed in BHKcells, or control media from cells infected with parental adenovirus(Zpar). The CM was concentrated 10-fold using a 15 ml centrifugal filterdevice with a 10K membrane filter (Ultrafree®; Millipore Corp., Bedford,Mass.), then diluted back to 3× with ITS medium and added to the cells.The control CM was generated from HaCaT cells infected with a parentalgreen fluorescent protein-expressing adenovirus and treated identicallyto the zvegf3 CM. Purified protein in a buffer containing 0.1% BSA wasserially diluted into ITS medium at concentrations of 1 μg/ml to 1 ng/mland added to the test plate. A control buffer of 0.1% BSA was dilutedidentically to the highest concentration of zvegf3 protein and added tothe plate. For measurement of [³H]thymidine incorporation, 20 μl of a 50μCi/ml stock in DMEM was added directly to the cells, for a finalactivity of 1 μCi/well. After another 24 hour incubation, mitogenicactivity was assessed by measuring the uptake of [³H]thymidine. Mediawere removed and cells were incubated with 0.1 ml of trypsin until cellsdetached. Cells were harvested onto 96-well filter plates using a sampleharvester (FilterMate™ harvester; Packard Instrument Co., Meriden,Conn.). The plates were then dried at 65° C. for 15 minutes, sealedafter adding 40 μl/well scintillation cocktail (Microscint™ O; PackardInstrument Co.) and counted on a microplate scintillation counter(Topcount®; Packard Instrument Co.)

Results presented in Table 8 demonstrate that zvegf3 CM hadapproximately 1.5-fold higher mitogenic activity on HAoSM cells overcontrol CM, and purified protein caused a maximal 1.8-fold increase in[³H]thymidine incorporation over the buffer control.

TABLE 8 CPM Incorporated Sample Mean St. dev. zvegf3 (3x CM) 81089 8866Zpar (3x CM) 58760 2558 zvegf3 GF domain, 1 μg/ml 63884 3281 zvegf3 GFdomain, 500 ng/ml 57484 9744 zvegf3 GF domain, 100 ng/ml 70844 10844 zvegf3 GF domain, 50 ng/ml 61164 2813 zvegf3 GF domain, 10 ng/ml 606761514 zvegf3 GE domain, 5 ng/ml 60197 2481 zvegf3 GF domain, 1 ng/ml49205 5208 Buffer control 39645 9793 PDGF 10 ng/ml (maximal 50634 4238response) Media alone (basal response) 24220 2463

Results presented in Table 9 demonstrate that zvegf3 CM had no mitogenicactivity on HUVEC compared to the control CM, and purified proteincaused a maximal 1.3-fold increase in [³H]thymidine incorporation overthe buffer control.

TABLE 9 CPM Incorporated Sample Mean St. dev. zvegf3 (3x CM) 6272310716  Zpar (3x CM) 61378 1553 zvegf3 VEGF domain, 1 μg/ml 44901 6592zvegf3 VEGF domain, 500 ng/ml 41921 5330 zvegf3 VEGF domain, 100 ng/ml35613 5187 zvegf3 VEGF domain, 50 ng/ml 31107  525 zvegf3 VEGE domain,10 ng/ml 28505 2950 zvegf3 VEGF domain, 5 ng/ml 29290  988 zvegf3 VEGFdomain, 1 ng/ml 28586 2718 Buffer control 33461  404 VEGF 50 ng/ml(maximal 53225 5229 response) Media alone (basal response) 22264 2814

Example 22

Recombinant zvegf3 protein was assayed for stimulation of intracellularcalcium release as an indicator of receptor binding and activation.Cells were cultured in chambered borosilicate coverglass slides. On theday of assay, cells were incubated for 30 minutes at room temperature inKRW buffer (KrebsRingerWollheim; 140 mM NaCl, 3.6 mM KCl, 0.5 mMNaH₂PO₄, 0.5 mM MgSO₄ 2 mM NaHCO₃, 3 mM glucose, 1.5 mM CaCl₂, 10 mMHEPES pH 7.4) containing 2 μM fura-2 AM (obtained from Molecular ProbesInc., Eugene, Oreg.), washed twice with KRW buffer, and allowed to sitat room temperature for at least 15 minutes before addition of growthfactor or cell-conditioned culture medium (CM) to be tested. Changes incytosolic calcium were measured by fluorescence ratio imaging(excitation at 340 nm divided by excitation at 380 nm). Digital imagingwas carried out using an inverted fluorescent microscope (Nikon TE300)equipped with an oil objective (Nikon 40×Plan Fluor). Images wereacquired using a Princeton CCD digital camera and analyzed withUniversal Imaging Metafluor software. Data are presented in Table 10.

TABLE 10 Cell Line Zvegf3 CM Control CM VEGF PDGF BB aortic ring cells +− − + pericytes + − − + aortic smooth muscle cells + − − + aorticadventitial fibroblasts + − − +

Example 23

Northern blot analysis was performed using total RNA from the humanneuronal and glial cell lines A172 (glioblastoma), NTera 2(teratocarcinoma neuronal precursor; obtained from Strategene CloningSystems, La Jolla, Calif.), U-87 MG (glioblastoma/astrocytoma), U-118 MG(glioblastoma), U138 MG (glioblastoma), U373 MG (glioblastoma). Exceptas noted, cell lines were obtained from American Type CultureCollection, Manassas, Va. Blots were prepared using 10 μg of RNA perlane. An approximately 400 bp DNA probe was generated by digestion ofhuman zvegf3 cDNA with EcoRI and BglII. The DNA probe was gelelectrophoresis followed by extraction using a spin column containing asilica gel membrane (QIAquick™ Gel Extraction Kit; Qiagen, Inc.,Valencia, Calif.). The probe was radioactively labeled with ³²P using acommercially available kit (Rediprime™ II random-prime labeling system;Amersham Corp., Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a push column.Hybridization took place overnight at 65° C. in a commercially availablesolution (ExpressHyb™ Hybridization Solution; Clontech Laboratories,Inc.).

The blots were then washed 4× in 2×SCC and 0.05% SDS at roomtemperature, followed by two washes in 0.1×SSC and 0.1% SDS at 50° C.One transcript size was detected at approximately 4 kb in A172, U-87 MG,U-118 MG, U138 MG, and U373 MG samples. Signal intensity was highest forU373 MG, U-118MG, and U-87 MG.

Example 24

10 μg of recombinant zvegf3 growth factor domain protein was combinedwith 438 μl PBS containing 1 mCi Na-¹²⁵I (Amersham Corp.). Onederivatized, nonporous polystyrene bead (IODO-Beads®; Pierce ChemicalCo., Rockford, Ill.) was added, and the reaction mixture was incubatedone minute on ice. The iodinated protein was separated fromunicorporated ¹²⁵I by gel filtration, elution buffer PBS, 0.25% gelatin.The active fraction contained 4.9 μg/mL ¹²⁵I-zvegf3 with a specificactivity of 4.3×10⁴ cpm/ng.

The following cell lines were plated into the wells of a 24-well tissueculture dish and cultured in growth medium for three days:

1. Rat aortic ring pool (ARC)

2. Rat aortic ring clone 14B (ARC#14B)

3. Human umbilical vien endothelial cells, passage 5 (HUVEC)

4. Human aortic adventicial fibroblasts, passage 4 (AOAF)

5. Human aortic smooth muscle cells, passage 9 (AOSMC)

6. Human retinal pericytes, passage 4 (pericytes)

Cells were washed once with ice cold binding buffer (RPMI containing0.1% BSA, 20 mM Tris:HCl, pH 7.2) and then 250 μl of the followingsolutions were added to each of three wells of the culture dishescontaining the test cells.

Binding solutions were prepared in 5 mL of binding buffer with 10 ng/mL¹²⁵I-zvegf3 and:

1. No addition.

2. One μg/mL zvegf 3.

3. One μg/mL VEGF (R&D Systems, Minneapolis, Minn.).

4. One μg/mL PDGF-BB.

5. Five μg/mL PDGF receptor α (R&D Systems).

6. Five μg/mL PDGF receptor β (as IgG Fc-receptor extracellular domainfusion).

The reaction mixtures were incubated on ice for 2 hours, then washedthree times with one mL of ice-cold binding buffer. The bound¹²⁵I-zvegf3 was quantitated by gamma counting a NaOH extract of thecells.

Results, shown in FIG. 5, indicate binding of zvegf3 to the PDGFreceptor α. The data are graphed as ¹²⁵I-zvegf3 bound/well. The errorbars represent standard deviations.

The experiment was repeated with the addition of rat liver stellatecells, passage 6 (Greenwel et al., Laboratory Investigation 69:210-216,1993). Stellate cells bound zvegf3 at a level comparable to pericytes.

Example 25

Binding of recombinant zvegf3 to PDGF alpha and beta receptors wasmeasured by mass spectrometry using a surface-enhanced laser desorptionand ionization (SELDI) instrument (ProteinChip™, Ciphergen Biosystems,Palo Alto, Calif.). For this experiment an 8-spot, preactivated surfacearray was used. To this amine-activated chip, protein-A (ZymedLaboratories, Inc., San Francisco, Calif.) was added at a concentrationof 1 mg/ml, and the chip was incubated at 4° C. for four hours. Afterblocking with 1M ethanolamine pH 8.0 and subsequent washes (once in 0.1%Triton X-100 in PBS; once in 100 mM Na Acetate, pH4.5, 0.5 M NaCl; oncein 100 mM Tris-HCl, pH8.5, 0.5 M NaCl; once in PBS), IgG Fc-receptorextracellular domain fusion proteins (PDGF alpha receptor, PDGF betareceptor, or unrelated control receptor) were added, and the chip wasincubated at 4° C. overnight. After three washes in PBS, 250 μl ofzvegf3 (300 ng/ml), PDGF-AA, or PDGF-BB was added, and the chip wasincubated overnight at 4° C. The chip was washed twice with 0.05% TritonX100, 100 mM HEPES pH 7.2, then twice with deionized water. The chip wasallowed to dry at room temperature before two additions of 0.3microliters of sinapinic acid (Ciphergen Biosystems) in a 50:50 mixtureof acetonitrile and 1% trifluroacetic acid. Ligands that bound receptorwere retained on the chip after washing and subsequently detected bymass spectrometry. Assignment of a + or − for binding was made bycomparing the PDGF receptor mass spectrometry profile to that of an Fconly control for each ligand. Data are shown in Table 11.

TABLE 11 PDGF AA PDGF AB PDGF BB ZVEGF3 PDGFR-alpha/Fc + + + +PDGFR-beta/Fc +/− +/− + −

Example 26

Northern blot analysis was performed with poly(A) RNA from humanvascular cell lines HUVEC (human umbilical vein endothelial cells;Cascade Biologics, Inc., Portland, Oreg.), HPAEC (human pulmonary arteryendothelial cells; Cascade Biologics), HAEC (human aortic endothelialcells; Cascade Biologics), AOSMC (aortic smooth muscle cells; CloneticsCorporation, Walkersville, Md.), UASMC (umbilical artery smooth musclecells; Clonetics Corp.), HISM (human intestinal smooth muscle; AmericanType Culture Collection, CRL 7130), SK5 (human dermal fibroblast cells;obtained from Dr. Russel Ross, University of Washington), NHLF (normalhuman lung fibroblasts; Clonetics Corp.), NHDF-neo (normal human dermalfibroblast-neonatal; Clonetics Corp.); and from leukemia cell linesDaudi, Raji, Molt-4, K562 (all obtained from Clontech Laboratories,Inc.), HL60, Jurkat, and Hut 78. RNA was loaded at 2 μg per lane. Anapproximately 490 bp DNA probe was generated by digestion of afull-length zVEGF3 clone with PvuI and StuI. The DNA probe waselectrophoresed and purified using a spin column containing a silica gelmembrane (QIAquick™ Gel Extraction Kit; Qiagen, Inc., Valencia, Calif.).The probe was radioactively labeled with ³²P using a commerciallyavailable kit (Rediprime™ II random-prime labeling system; AmershamCorp., Arlington Heights, Ill.) according to the manufacturer'sspecifications. The probe was purified using a push column. Expresshyb(Clontech, Palo Alto, Calif.) solution was used for the hybridizingsolution for the blots. Hybridization took place overnight at 65° C. ina commercially available hybridization solution (ExpressHyb™Hybridization Solution; Clontech Laboratories, Inc., Palo Alto, Calif.).The blots were then washed four times in 2×SCC and 0.1% SDS at roomtemperature, followed by two washes in 0.1×SSC and 0.1% SDS at 50° C.One transcript size was detected at approximately 4 kb in NHLF,NHDF-neo, SK5, UASMC, HAEC, AoSMC, Jurkat, Hut78. Signal intensity washighest for UASMC, SK5, NHLF, and NHDF-neo.

Example 27

The effects of zvegf3 on vascular endothelial regeneration and intimalhyperplasia are tested in a balloon injured rat carotid artery model.Adenovirus is used as a delivery vector.

To determine the infectivity of adenovirus and the level of geneexpression obtained in the vascular wall, rats are balloon injured asdisclosed below and infused with an adenovirus vector comprising anexpression unit for green fluourescent protein (GFP). Three groups ofthree rats each are infused with doses of 1.5×10¹⁰ pfu/ml, 3×10¹⁰pfu/ml, and 6×10¹⁰ pfu/ml. Injured and uninjured carotids are harvested48 hours after infection and fixed in 10% buffered formalin for 24 hrs.The tissue is processed and analyzed using an anti-GFP antibody todetermine % infectivity.

The effects of zvegf3 are determined in a 14-day study using the optimaldose determined from the GFP study. Two groups of 14 animals each areballoon-injured and infected with either zvegf3 adenovirus or controladenovirus. The left common carotid is isolated, and the flow of bloodthrough the vessel is stopped by tying off the internal carotid, theexternal carotid, and proximally, the common carotid. An arteriotomy ismade between the tie on the external carotid and the bifurcation, andthe vessel is rinsed out with lactated Ringer's. A 2F-embolectomycatheter is inserted, inflated, and removed, while twisting, to removeendothelial cells; this procedure is done three times. The vessel isthen rinsed again, and approximately 50 μl of adenovirus solution isinjected into it using a catheter of silastic tubing. The catheter istied into the vessel just distal to the bifurcation and left in placefor approximately 20 minutes. The catheter is then removed, and thevessel is flushed briefly with blood by loosening the proximal tie. Atie is made just distal to the bifurcation. Blood flow is restored byremoving the tie on the internal carotid and the proximal tie on thecommon carotid. The ties on the external carotid remain. To determinezvegf3 protein production in the vessel wall, two animals from eachgroup are sacrificed on days one and seven. Tissues are processed forimmunohistochemical analysis and Western blotting analysis. Forimmunohistochemical analysis, tissues are kept in formalin for 24 hours,then transferred to 70% ethyl alcohol. For Western blotting analysis,tissues are flash frozen and stored at −80° C. At thirteen days animalsare given BrdU tablets subcutaneously. At fourteen days (24 hours afterBrdU insertion) Evan's Blue dye is given intravenously to stain fornon-endothelialized segments, and the animals are bled and sacrificed.Animals are then exsanguinated and perfusion-fixed with 10% bufferedformalin. Both carotids, liver, kidney and spleen are harvested. Thecarotids are visually inspected, and re-endothelialization isquantitated by measuring the distance from the bifurcation to the distaldye (white/blue) boundary. All tissue are kept in formalin for 24 hoursthen transferred to 70% ethyl alcohol. The carotids are sectioned intothree pieces each and embedded in paraffin blocks. The liver, kidney,and spleen are visually inspected and processed. Slides are made ofcross-sections of the carotids and stained with Hematoxylin and Eosin,then measured using the SPOT® diagnostic program, (DiagnosticInstruments, Inc., Sterling Heights, Mich.). Measurements include thelength of the internal elastic lamina and the areas of the media,intima, and lumen. ICC analysis includes the number of infected cells(using anti-gfp antibodies), cell proliferation (BrdU labeling), and %cell death.

A third study is conducted to determine the time course of zvegf3 geneexpression following balloon injury. Carotids are harvested fromballoon-injured animals (5 animals/time point) at T=0 (non-injured), T=6hrs, 1, 4, 7, and 14 days. The carotids are flash-frozen and stored at−80° C. for Northern blot analysis.

Example 28

An expression plasmid containing a polynucleotide encoding human zvegf3fused N-terminally to maltose binding protein (MBP) was constructed viahomologous recombination.

A fragment of human zvegf3 cDNA was isolated using PCR. Two primers wereused in the production of the human zvegf3 fragment in a PCR reaction.Primer ZC20,572 (SEQ ID NO:44), contained 40 bp of vector flankingsequence and 25 bp corresponding to the amino terminus of human zvegf3,and primer ZC20,573 (SEQ ID NO:45) contained 40 bp of the 3′ endcorresponding to flanking vector sequence and 25 bp corresponding to thecarboxyl terminus of human zvegf3. The PCR reaction conditions were 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for1.5 minutes; followed by 4° C. soak, run in duplicate. A 2-μl aliquot ofthe 100-μl reaction mixture was run on a 1.0% agarose gel withTris/borate/EDTA buffer for analysis, and the expected band ofapproximately 1000 bp was seen. The remaining 90 μl of the reactionmixture was combined with the second PCR tube precipitated with 400 μlof absolute ethanol to be used for recombining into the Smal-cutrecipient vector pTAP98 to produce the construct encoding the MBP-zvegf3fusion.

Plasmid pTAP98 was derived from the plasmids pRS316 (a Saccharomycescerevisiae shuttle vector; see, Hieter and Sikorski, Genetics 122:19-27,1989) and pMAL™-c2X (New England Biolabs; Beverly, Mass.). The lattervector carries the tac promoter driving MalE (gene encoding MBP)followed by a His tag, a thrombin cleavage site, a cloning site, and therrnB terminator. The vector pTAP98 was constructed using yeasthomologous recombination. 100 ng of EcoRI-cut pMAL™-c2X was recombinedwith 1 μg PvuI-cut pRS316, 1 μg linker, and 1 μg Scal/EcoRI-cut pRS316.The linker was constructued by combining oligonucleotides ZC19,372 (SEQID NO:46) (100 pmole), ZC19,351 (SEQ ID NO:47) (1 pmole), ZC19,352 (SEQID NO:48) (1 pmole), and ZC19,371 (SEQ ID NO:49) (100 pmole) in a PCRreaction for 10 cycles of 94° C. for 30 seconds, 50° C. for 30 seconds,and 72° C. for 30 seconds; followed by 4° C. soak. PCR products wereconcentrated via 100% ethanol precipitation.

A vector containing the MBP-zvegf3 fusion sequence was constructed byhomologous recombination. One hundred microliters of competent yeastcells (S. cerevisiae) were combined with 10 μl of a mixture containingapproximately 1 μg of the human zvegf3 insert and 100 ng of SmaIdigested pTAP98 vector, and transferred to a 0.2-cm electroporationcuvette. The yeast/DNA mixture was electropulsed at 0.75 kV (5 kV/cm),infinite ohms, 25 μF. To each cuvette was added 600 μl of 1.2 Msorbitol. The yeast was then plated in two 300-μl aliquots onto two -URAD plates and incubated at 30° C. After about 48 hours, the Ura⁺ yeasttransformants from a single plate were resuspended in 1 ml H₂O and spunbriefly to pellet the yeast cells. The cell pellet was resuspended in 1ml of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH8.0, 1 mM EDTA). Five hundred microliters of the lysis mixture was addedto an Eppendorf tube containing 300 μl acid-washed glass beads and 200μl phenol-chloroform, vortexed for 1 minute intervals two or threetimes, followed by a 5 minute spin in a microcentrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA was precipitated with 600 μl ethanol (EtOH),then centrifuged for 10 minutes at 4° C. The DNA pellet was resuspendedin 100 μl H₂O. Electrocompetent E. coli cells (MC1061; Casadaban et.al., J. Mol. Biol. 138: 179-207) were transformed with 1 μl of the yeastDNA prep in a volume of 40 μl. The cells were electropulsed at 2.0 kV,25 μF, 400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto™Tryptone (Difco Laboratories, Detroit, Mich.), 0.5% yeast extract (DifcoLaboratories), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was plated in one aliquot on LB AMP plates (LB broth (Lennox),1.8% agar (Bacto™; Difco Laboratories), 100 mg/L. ampicillin).Individual clones harboring the correct expression construct for zvegf3were identified by expression. Cells were grown in minimal mediumsupplemented with casamino acids and 100 μg/ml of ampicillin overnight.50 μl of the overnight culture was used to inoculate 2 ml of freshmedium. Cultures were grown at 37° C., shaking for 2 hours. One ml ofthe culture was induced with 1 mM IPTG. 2-4 hours later 250 μl of eachculture was mixed with 250 μl acid-washed glass beads and 250 μl Thornerbuffer (8 M urea, 100 mM Tris pH 7.0, 10% glycerol, 2 mM EDTA, 5% SDS)supplemented with 5% β-ME and dye. Samples were vortexed for one minuteand heated to 65° C. for 5-10 minutes. 20 μl was loaded per lane on a4%-12% PAGE gel (NOVEX, San Diego, Calif.). Gels were run in 1×MESbuffer. The positive clones were designated pCZR236 and subjected tosequence analysis. The polynucleotide sequence of the MBP-zvegf3 fusionin pCZR23G is shown in SEQ ID NO:50.

To express the fusion protein, 1 μl of sequencing DNA was used totransform E. coli strain W3110 (obtained from American Type CultureCollection, Manassas, Va.). The cells were electropulsed at 2.0 kV, 25μF and 400 ohms. Following electroporation, 0.6 ml SOC (2% Bacto™Tryptone (Difco Laboratories), 0.5% yeast extract (Difco Laboratories),10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose) wasplated in one aliquot on LB AMP plates (LB broth (Lennox), 1.8% Bacto™Agar (Difco Laboratories), 100 mg/L ampicillin). Cells were picked fromthe plate and grown in minimal medium containing casamino acidsovernight. A 50-μl aliquot of the overnight culture was used toinoculate 2 ml of fresh medium. Cultures were grown at 37° C. withshaking for 2 hours. One ml of the culture was induced with 1 mM IPTG,and the cells were lysed essentially as described above. Twenty-μlaliquots of the lysate were analyzed by gel electrophoresis as describedabove.

Example 29

Recombinant zvegf3 was analyzed for mitogenic activity on rat stellatecells (obtained from N. Fausto, University Of Washington). Stellatecells were plated at a density of 2,000 cells/well in 96-well cultureplates and grown for approximately 72 hours in DMEM containing 10% fetalcalf serum at 37° C. Cells were quiesced by incubating them for 20 hoursin serum-free DMEM/Ham's F-12 medium containing insulin (5 μg/ml),transferrin (20 μg/ml), and selenium (16 pg/ml) (ITS). At the time ofthe assay, the medium was removed, and test samples were added to thewells in triplicate. Test samples consisted of either conditioned media(CM) from adenovirally-infected HaCaT human keratinocyte cells (Boukampet al., J. Cell. Biol. 106:761-771, 1988) expressing full-length zvegf3,purified growth factor domain expressed in BHK cells, or control mediafrom cells infected with parental adenovirus (Zpar) containing anexpression unit for green fluorescent protein. The CM was concentrated10-fold using a 15-ml centrifugal filter device with a 10K membranefilter (Ultrafree®; Millipore Corp., Bedford, Mass.), then diluted backto 3× with ITS medium and added to the cells. Purified protein in abuffer containing 0.1% ABSA was serially diluted into ITS medium atconcentrations of 1 μg/ml to 1 ng/ml and added to the test plate. Acontrol buffer of 0.1% BSA was diluted identically to the highestconcentration of zvegf3 protein and added to the plate. For measurementof [³H]thymidine incorporation, 20 μl of a 50 μCi/ml stock in DMEM wasadded directly to the cells, for a final activity of 1 μCi/well. Afteranother 24-hour incubation, mitogenic activity was assessed by measuringthe uptake of [³H]thymidine. Media were removed, and cells wereincubated with 0.1 ml of trypsin until cells detached. Cells wereharvested onto 96-well filter plates using a sample harvester(FilterMate™ harvester; Packard Instrument Co., Meriden, Conn.). Theplates were then dried at 65° C. for 15 minutes, sealed after adding 40μl/well scintillation cocktail (Microscint™ O; Packard Instrument Co.)and counted on a microplate scintillation counter (Topcount®; PackardInstrument Co.).

Results, presented in Table 12, demonstrated that zvegf3 CM hadapproximately 4.4-fold higher mitogenic activity on stellate cells overcontrol CM, and purified protein at 100 ng/ml caused a maximal 14-foldincrease in [³H]thymidine incorporation over the buffer control.

TABLE 12 CPM Incorporated Sample Mean St. dev. zvegf3 (2x CM) 42489 1306Zpar (2x CM)  9629  540 zvegf3 GF domain, 100 ng/ml 77540 4142 zvegf3 GFdomain, 33.3 ng/ml 74466 18142  zvegf3 GF domain, 11.1 ng/ml 52462 6239zvegf3 GF domain, 3.7 ng/ml 15128 4989 Buffer control  5618  573 PDGF-BB20 ng/ml 19741 2075 PDGF-AA 20 ng/ml 33133 3325 Media alone (basalresponse)  6765  226

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 50 <210> SEQ ID NO 1 <211> LENGTH: 1760<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (154)...(1191) <400> SEQUENCE: 1attatgtgga aactaccctg cgattctctg ctgccagagc aggctcggcg ct#tccacccc     60agtgcagcct tcccctggcg gtggtgaaag agactcggga gtcgctgctt cc#aaagtgcc    120 cgccgtgagt gagctctcac cccagtcagc caa atg agc ctc tt#c ggg ctt ctc     174                    #                  # Met Ser Leu Phe Gly Leu Leu                    #                  #  1               5 ctg ctg aca tct gcc ctg gcc ggc cag aga ca#g ggg act cag gcg gaa      222Leu Leu Thr Ser Ala Leu Ala Gly Gln Arg Gl #n Gly Thr Gln Ala Glu         10          #         15          #         20tcc aac ctg agt agt aaa ttc cag ttt tcc ag#c aac aag gaa cag aac      270Ser Asn Leu Ser Ser Lys Phe Gln Phe Ser Se #r Asn Lys Glu Gln Asn     25              #     30              #     35gga gta caa gat cct cag cat gag aga att at#t act gtg tct act aat      318Gly Val Gln Asp Pro Gln His Glu Arg Ile Il #e Thr Val Ser Thr Asn 40                  # 45                  # 50                  # 55gga agt att cac agc cca agg ttt cct cat ac#t tat cca aga aat acg      366Gly Ser Ile His Ser Pro Arg Phe Pro His Th #r Tyr Pro Arg Asn Thr                 60  #                 65  #                 70gtc ttg gta tgg aga tta gta gca gta gag ga#a aat gta tgg ata caa      414Val Leu Val Trp Arg Leu Val Ala Val Glu Gl #u Asn Val Trp Ile Gln             75      #             80      #             85ctt acg ttt gat gaa aga ttt ggg ctt gaa ga#c cca gaa gat gac ata      462Leu Thr Phe Asp Glu Arg Phe Gly Leu Glu As #p Pro Glu Asp Asp Ile         90          #         95          #        100tgc aag tat gat ttt gta gaa gtt gag gaa cc#c agt gat gga act ata      510Cys Lys Tyr Asp Phe Val Glu Val Glu Glu Pr #o Ser Asp Gly Thr Ile    105               #   110               #   115tta ggg cgc tgg tgt ggt tct ggt act gta cc#a gga aaa cag att tct      558Leu Gly Arg Trp Cys Gly Ser Gly Thr Val Pr #o Gly Lys Gln Ile Ser120                 1 #25                 1 #30                 1 #35aaa gga aat caa att agg ata aga ttt gta tc#t gat gaa tat ttt cct      606Lys Gly Asn Gln Ile Arg Ile Arg Phe Val Se #r Asp Glu Tyr Phe Pro                140   #               145   #               150tct gaa cca ggg ttc tgc atc cac tac aac at#t gtc atg cca caa ttc      654Ser Glu Pro Gly Phe Cys Ile His Tyr Asn Il #e Val Met Pro Gln Phe            155       #           160       #           165aca gaa gct gtg agt cct tca gtg cta ccc cc#t tca gct ttg cca ctg      702Thr Glu Ala Val Ser Pro Ser Val Leu Pro Pr #o Ser Ala Leu Pro Leu        170           #       175           #       180gac ctg ctt aat aat gct ata act gcc ttt ag#t acc ttg gaa gac ctt      750Asp Leu Leu Asn Asn Ala Ile Thr Ala Phe Se #r Thr Leu Glu Asp Leu    185               #   190               #   195att cga tat ctt gaa cca gag aga tgg cag tt#g gac tta gaa gat cta      798Ile Arg Tyr Leu Glu Pro Glu Arg Trp Gln Le #u Asp Leu Glu Asp Leu200                 2 #05                 2 #10                 2 #15tat agg cca act tgg caa ctt ctt ggc aag gc#t ttt gtt ttt gga aga      846Tyr Arg Pro Thr Trp Gln Leu Leu Gly Lys Al #a Phe Val Phe Gly Arg                220   #               225   #               230aaa tcc aga gtg gtg gat ctg aac ctt cta ac#a gag gag gta aga tta      894Lys Ser Arg Val Val Asp Leu Asn Leu Leu Th #r Glu Glu Val Arg Leu            235       #           240       #           245tac agc tgc aca cct cgt aac ttc tca gtg tc#c ata agg gaa gaa cta      942Tyr Ser Cys Thr Pro Arg Asn Phe Ser Val Se #r Ile Arg Glu Glu Leu        250           #       255           #       260aag aga acc gat acc att ttc tgg cca ggt tg#t ctc ctg gtt aaa cgc      990Lys Arg Thr Asp Thr Ile Phe Trp Pro Gly Cy #s Leu Leu Val Lys Arg    265               #   270               #   275tgt ggt ggg aac tgt gcc tgt tgt ctc cac aa#t tgc aat gaa tgt caa     1038Cys Gly Gly Asn Cys Ala Cys Cys Leu His As #n Cys Asn Glu Cys Gln280                 2 #85                 2 #90                 2 #95tgt gtc cca agc aaa gtt act aaa aaa tac ca#c gag gtc ctt cag ttg     1086Cys Val Pro Ser Lys Val Thr Lys Lys Tyr Hi #s Glu Val Leu Gln Leu                300   #               305   #               310aga cca aag acc ggt gtc agg gga ttg cac aa#a tca ctc acc gac gtg     1134Arg Pro Lys Thr Gly Val Arg Gly Leu His Ly #s Ser Leu Thr Asp Val            315       #           320       #           325gcc ctg gag cac cat gag gag tgt gac tgt gt#g tgc aga ggg agc aca     1182Ala Leu Glu His His Glu Glu Cys Asp Cys Va #l Cys Arg Gly Ser Thr        330           #       335           #       340gga gga tag ccgcatcacc accagcagct cttgcccaga gctgtgcag#t             1231 Gly Gly  *     345gcagtggctg attctattag agaacgtatg cgttatctcc atccttaatc tc#agttgttt   1291gcttcaagga cctttcatct tcaggattta cagtgcattc tgaaagagga ga#catcaaac   1351agaattagga gttgtgcaac agctcttttg agaggaggcc taaaggacag ga#gaaaaggt   1411cttcaatcgt ggaaagaaaa ttaaatgttg tattaaatag atcaccagct ag#tttcagag   1471ttaccatgta cgtattccac tagctgggtt ctgtatttca gttctttcga ta#cggcttag   1531ggtaatgtca gtacaggaaa aaaactgtgc aagtgagcac ctgattccgt tg#ccttgctt   1591aactctaaag ctccatgtcc tgggcctaaa atcgtataaa atctggattt tt#tttttttt   1651tttttgctca tattcacata tgtaaaccag aacattctat gtactacaaa cc#tggttttt   1711 aaaaaggaac tatgttgcta tgaattaaac ttgtgtcgtg ctgatagga  #             1760 <210> SEQ ID NO 2 <211> LENGTH: 345 <212> TYPE: PRT<213> ORGANISM: Homo sapiens <400> SEQUENCE: 2Met Ser Leu Phe Gly Leu Leu Leu Leu Thr Se #r Ala Leu Ala Gly Gln 1               5   #                10   #                15Arg Gln Gly Thr Gln Ala Glu Ser Asn Leu Se #r Ser Lys Phe Gln Phe            20       #            25       #            30Ser Ser Asn Lys Glu Gln Asn Gly Val Gln As #p Pro Gln His Glu Arg        35           #        40           #        45Ile Ile Thr Val Ser Thr Asn Gly Ser Ile Hi #s Ser Pro Arg Phe Pro    50               #    55               #    60His Thr Tyr Pro Arg Asn Thr Val Leu Val Tr #p Arg Leu Val Ala Val65                   #70                   #75                   #80Glu Glu Asn Val Trp Ile Gln Leu Thr Phe As #p Glu Arg Phe Gly Leu                85   #                90   #                95Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr As #p Phe Val Glu Val Glu            100       #           105       #           110Glu Pro Ser Asp Gly Thr Ile Leu Gly Arg Tr #p Cys Gly Ser Gly Thr        115           #       120           #       125Val Pro Gly Lys Gln Ile Ser Lys Gly Asn Gl #n Ile Arg Ile Arg Phe    130               #   135               #   140Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gl #y Phe Cys Ile His Tyr145                 1 #50                 1 #55                 1 #60Asn Ile Val Met Pro Gln Phe Thr Glu Ala Va #l Ser Pro Ser Val Leu                165   #               170   #               175Pro Pro Ser Ala Leu Pro Leu Asp Leu Leu As #n Asn Ala Ile Thr Ala            180       #           185       #           190Phe Ser Thr Leu Glu Asp Leu Ile Arg Tyr Le #u Glu Pro Glu Arg Trp        195           #       200           #       205Gln Leu Asp Leu Glu Asp Leu Tyr Arg Pro Th #r Trp Gln Leu Leu Gly    210               #   215               #   220Lys Ala Phe Val Phe Gly Arg Lys Ser Arg Va #l Val Asp Leu Asn Leu225                 2 #30                 2 #35                 2 #40Leu Thr Glu Glu Val Arg Leu Tyr Ser Cys Th #r Pro Arg Asn Phe Ser                245   #               250   #               255Val Ser Ile Arg Glu Glu Leu Lys Arg Thr As #p Thr Ile Phe Trp Pro            260       #           265       #           270Gly Cys Leu Leu Val Lys Arg Cys Gly Gly As #n Cys Ala Cys Cys Leu        275           #       280           #       285His Asn Cys Asn Glu Cys Gln Cys Val Pro Se #r Lys Val Thr Lys Lys    290               #   295               #   300Tyr His Glu Val Leu Gln Leu Arg Pro Lys Th #r Gly Val Arg Gly Leu305                 3 #10                 3 #15                 3 #20His Lys Ser Leu Thr Asp Val Ala Leu Glu Hi #s His Glu Glu Cys Asp                325   #               330   #               335Cys Val Cys Arg Gly Ser Thr Gly Gly             340      #           345 <210> SEQ ID NO 3 <211> LENGTH: 116 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: peptide motif <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (2)...(19)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (20)...(34)<223> OTHER INFORMATION: Xaa is any amino acid  #or not present<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (36)...(36)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (38)...(38)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (40)...(45)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (46)...(72)<223> OTHER INFORMATION: Xaa is any amino acid  #or not present<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (74)...(93)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (94)...(113)<223> OTHER INFORMATION: Xaa is any amino acid  #not present<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (115)...(115)<223> OTHER INFORMATION: Xaa is any amino acid <400> SEQUENCE: 3Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa 1               5   #                10   #                15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa            20       #            25       #            30Xaa Xaa Cys Xaa Gly Xaa Cys Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa        35           #        40           #        45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa    50               #    55               #    60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xa #a Xaa Xaa Xaa Xaa Xaa65                   #70                   #75                   #80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa                85   #                90   #                95Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa            100       #           105       #           110Xaa Cys Xaa Cys         115 <210> SEQ ID NO 4 <211> LENGTH: 24<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: peptide motif <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (2)...(2)<223> OTHER INFORMATION: Xaa is Lys or Arg <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (4)...(4)<223> OTHER INFORMATION: Xaa is Asp, Asn or  #Glu <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (5)...(5)<223> OTHER INFORMATION: Xaa is Trp, Tyr or  #Phe <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (6)...(16)<223> OTHER INFORMATION: Xaa is any amino acid <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (17)...(20)<223> OTHER INFORMATION: Xaa is any amino acid  #or not present<220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (22)...(22)<223> OTHER INFORMATION: Xaa is Lys or Arg <220> FEATURE:<221> NAME/KEY: VARIANT <222> LOCATION: (23)...(23)<223> OTHER INFORMATION: Xaa is Trp, Tyr or  #Phe <400> SEQUENCE: 4Cys Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa #a Xaa Xaa Xaa Xaa Xaa 1               5   #                10   #                15Xaa Xaa Xaa Xaa Gly Xaa Xaa Cys             20 <210> SEQ ID NO 5<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: peptide tag <400> SEQUENCE: 5Glu Tyr Met Pro Met Glu  1               5 <210> SEQ ID NO 6<211> LENGTH: 1035 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: degenerate sequence derived #from SEQ ID NOS: 1       and 2 <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(1035)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 6atgwsnytnt tyggnytnyt nytnytnacn wsngcnytng cnggncarmg nc#arggnacn     60cargcngarw snaayytnws nwsnaartty carttywsnw snaayaarga rc#araayggn    120gtncargayc cncarcayga rmgnathath acngtnwsna cnaayggnws na#thcaywsn    180ccnmgnttyc cncayacnta yccnmgnaay acngtnytng tntggmgnyt ng#tngcngtn    240gargaraayg tntggathca rytnacntty gaygarmgnt tyggnytnga rg#ayccngar    300gaygayatht gyaartayga yttygtngar gtngargarc cnwsngaygg na#cnathytn    360ggnmgntggt gyggnwsngg nacngtnccn ggnaarcara thwsnaargg na#aycarath    420mgnathmgnt tygtnwsnga ygartaytty ccnwsngarc cnggnttytg ya#thcaytay    480aayathgtna tgccncartt yacngargcn gtnwsnccnw sngtnytncc nc#cnwsngcn    540ytnccnytng ayytnytnaa yaaygcnath acngcnttyw snacnytnga rg#ayytnath    600mgntayytng arccngarmg ntggcarytn gayytngarg ayytntaymg nc#cnacntgg    660carytnytng gnaargcntt ygtnttyggn mgnaarwsnm gngtngtnga yy#tnaayytn    720ytnacngarg argtnmgnyt ntaywsntgy acnccnmgna ayttywsngt nw#snathmgn    780gargarytna armgnacnga yacnathtty tggccnggnt gyytnytngt na#armgntgy    840ggnggnaayt gygcntgytg yytncayaay tgyaaygart gycartgygt nc#cnwsnaar    900gtnacnaara artaycayga rgtnytncar ytnmgnccna aracnggngt nm#gnggnytn    960cayaarwsny tnacngaygt ngcnytngar caycaygarg artgygaytg yg#tntgymgn   1020 ggnwsnacng gnggn               #                  #                   #  1035 <210> SEQ ID NO 7 <211> LENGTH: 17<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 7mgntgyggng gnaaytg              #                   #                  #   17 <210> SEQ ID NO 8 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 8mgntgydsng gnwrytg              #                   #                  #   17 <210> SEQ ID NO 9 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 9carywnccns hrcanck              #                   #                  #   17 <210> SEQ ID NO 10 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 10ttytggccng gntgyyt              #                   #                  #   17 <210> SEQ ID NO 11 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 11ntnddnccnn sntgybt              #                   #                  #   17 <210> SEQ ID NO 12 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 12avrcansnng gnhhnan              #                   #                  #   17 <210> SEQ ID NO 13 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 13caygargart gygaytg              #                   #                  #   17 <210> SEQ ID NO 14 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 14caynnnnvnt gyvvntg              #                   #                  #   17 <210> SEQ ID NO 15 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 15canbbrcanb nnnnrtg              #                   #                  #   17 <210> SEQ ID NO 16 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 16tgyacnccnm gnaaytt              #                   #                  #   17 <210> SEQ ID NO 17 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 17tgyhnnmcnm knrmndh              #                   #                  #   17 <210> SEQ ID NO 18 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 18dhnkynmkng knndrca              #                   #                  #   17 <210> SEQ ID NO 19 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 19tgyaartayg aytwygt              #                   #                  #   17 <210> SEQ ID NO 20 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 20acrwartcrt ayttrca              #                   #                  #   17 <210> SEQ ID NO 21 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 21ywnggnmrnt dbtgygg              #                   #                  #   17 <210> SEQ ID NO 22 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 22ccrcavhany knccnwr              #                   #                  #   17 <210> SEQ ID NO 23 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 23tdbccnmand vntaycc              #                   #                  #   17 <210> SEQ ID NO 24 <211> LENGTH: 17 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(17)<223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 24ggrtanbhnt knggvha              #                   #                  #   17 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 25agcaggtcca gtggcaaagc             #                  #                   # 20 <210> SEQ ID NO 26 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 26cgtttgatga aagatttggg c            #                  #                   #21 <210> SEQ ID NO 27 <211> LENGTH: 21<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 27ggaggtctat ataagcagag c            #                  #                   #21 <210> SEQ ID NO 28 <211> LENGTH: 18<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 28taacagagga ggtaagat              #                   #                  #  18 <210> SEQ ID NO 29 <211> LENGTH: 18 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 29tcggttctct ttagttct              #                   #                  #  18 <210> SEQ ID NO 30 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 30tctggacgtc ctcctgctgg tatag           #                  #               25 <210> SEQ ID NO 31 <211> LENGTH: 25 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 31ggtatggagc caggggcaag ttggg           #                  #               25 <210> SEQ ID NO 32 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 32gagtggcaac ttccagggcc aggagag           #                  #             27 <210> SEQ ID NO 33 <211> LENGTH: 27 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer <400> SEQUENCE: 33cttttgctag cctcaaccct gactatc           #                  #             27 <210> SEQ ID NO 34 <211> LENGTH: 35 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC20, #180<400> SEQUENCE: 34 cgcgcggttt aaacgccacc atgagcctct tcggg       #                   #       35 <210> SEQ ID NO 35 <211> LENGTH: 32<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Oligonucleotide primer ZC20, #181<400> SEQUENCE: 35 cgtatcggcg cgccctatcc tcctgtgctc cc       #                   #          32 <210> SEQ ID NO 36 <211> LENGTH: 1882<212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (226)...(1338) <400> SEQUENCE: 36ccgtcaccat ttatcagctc agcaccacaa ggaagtgcgg cacccacacg cg#ctcggaaa     60gttcagcatg caggaagttt ggggagagct cggcgattag cacagcgacc cg#ggccagcg    120cagggcgagc gcaggcggcg agagcgcagg gcggcgcggc gtcggtcccg gg#agcagaac    180 ccggcttttt cttggagcga cgctgtctct agtcgctgat cccaa atg #cac cgg ctc    237                    #                  #              Met His A #rg Leu                    #                  #               1 atc ttt gtc tac act cta atc tgc gca aac tt#t tgc agc tgt cgg gac      285Ile Phe Val Tyr Thr Leu Ile Cys Ala Asn Ph #e Cys Ser Cys Arg Asp 5                  #  10                 #  15                 #  20act tct gca acc ccg cag agc gca tcc atc aa#a gct ttg cgc aac gcc      333Thr Ser Ala Thr Pro Gln Ser Ala Ser Ile Ly #s Ala Leu Arg Asn Ala                 25  #                 30  #                 35aac ctc agg cga gat gag agc aat cac ctc ac#a gac ttg tac cga aga      381Asn Leu Arg Arg Asp Glu Ser Asn His Leu Th #r Asp Leu Tyr Arg Arg             40      #             45      #             50gat gag acc atc cag gtg aaa gga aac ggc ta#c gtg cag agt cct aga      429Asp Glu Thr Ile Gln Val Lys Gly Asn Gly Ty #r Val Gln Ser Pro Arg         55          #         60          #         65ttc ccg aac agc tac ccc agg aac ctg ctc ct#g aca tgg cgg ctt cac      477Phe Pro Asn Ser Tyr Pro Arg Asn Leu Leu Le #u Thr Trp Arg Leu His     70              #     75              #     80tct cag gag aat aca cgg ata cag cta gtg tt#t gac aat cag ttt gga      525Ser Gln Glu Asn Thr Arg Ile Gln Leu Val Ph #e Asp Asn Gln Phe Gly 85                  # 90                  # 95                  #100tta gag gaa gca gaa aat gat atc tgt agg ta#t gat ttt gtg gaa gtt      573Leu Glu Glu Ala Glu Asn Asp Ile Cys Arg Ty #r Asp Phe Val Glu Val                105   #               110   #               115gaa gat ata tcc gaa acc agt acc att att ag#a gga cga tgg tgt gga      621Glu Asp Ile Ser Glu Thr Ser Thr Ile Ile Ar #g Gly Arg Trp Cys Gly            120       #           125       #           130cac aag gaa gtt cct cca agg ata aaa tca ag#a acg aac caa att aaa      669His Lys Glu Val Pro Pro Arg Ile Lys Ser Ar #g Thr Asn Gln Ile Lys        135           #       140           #       145atc aca ttc aag tcc gat gac tac ttt gtg gc#t aaa cct gga ttc aag      717Ile Thr Phe Lys Ser Asp Asp Tyr Phe Val Al #a Lys Pro Gly Phe Lys    150               #   155               #   160att tat tat tct ttg ctg gaa gat ttc caa cc#c gca gca gct tca gag      765Ile Tyr Tyr Ser Leu Leu Glu Asp Phe Gln Pr #o Ala Ala Ala Ser Glu165                 1 #70                 1 #75                 1 #80acc aac tgg gaa tct gtc aca agc tct att tc#a ggg gta tcc tat aac      813Thr Asn Trp Glu Ser Val Thr Ser Ser Ile Se #r Gly Val Ser Tyr Asn                185   #               190   #               195tct cca tca gta acg gat ccc act ctg att gc#g gat gct ctg gac aaa      861Ser Pro Ser Val Thr Asp Pro Thr Leu Ile Al #a Asp Ala Leu Asp Lys            200       #           205       #           210aaa att gca gaa ttt gat aca gtg gaa gat ct#g ctc aag tac ttc aat      909Lys Ile Ala Glu Phe Asp Thr Val Glu Asp Le #u Leu Lys Tyr Phe Asn        215           #       220           #       225cca gag tca tgg caa gaa gat ctt gag aat at#g tat ctg gac acc cct      957Pro Glu Ser Trp Gln Glu Asp Leu Glu Asn Me #t Tyr Leu Asp Thr Pro    230               #   235               #   240cgg tat cga ggc agg tca tac cat gac cgg aa#g tca aaa gtt gac ctg     1005Arg Tyr Arg Gly Arg Ser Tyr His Asp Arg Ly #s Ser Lys Val Asp Leu245                 2 #50                 2 #55                 2 #60gat agg ctc aat gat gat gcc aag cgt tac ag#t tgc act ccc agg aat     1053Asp Arg Leu Asn Asp Asp Ala Lys Arg Tyr Se #r Cys Thr Pro Arg Asn                265   #               270   #               275tac tcg gtc aat ata aga gaa gag ctg aag tt#g gcc aat gtg gtc ttc     1101Tyr Ser Val Asn Ile Arg Glu Glu Leu Lys Le #u Ala Asn Val Val Phe            280       #           285       #           290ttt cca cgt tgc ctc ctc gtg cag cgc tgt gg#a gga aat tgt ggc tgt     1149Phe Pro Arg Cys Leu Leu Val Gln Arg Cys Gl #y Gly Asn Cys Gly Cys        295           #       300           #       305gga act gtc aac tgg agg tcc tgc aca tgc aa#t tca ggg aaa acc gtg     1197Gly Thr Val Asn Trp Arg Ser Cys Thr Cys As #n Ser Gly Lys Thr Val    310               #   315               #   320aaa aag tat cat gag gta tta cag ttt gag cc#t ggc cac atc aag agg     1245Lys Lys Tyr His Glu Val Leu Gln Phe Glu Pr #o Gly His Ile Lys Arg325                 3 #30                 3 #35                 3 #40agg ggt aga gct aag acc atg gct cta gtt ga#c atc cag ttg gat cac     1293Arg Gly Arg Ala Lys Thr Met Ala Leu Val As #p Ile Gln Leu Asp His                345   #               350   #               355cat gaa cga tgc gat tgt atc tgc agc tca ag#a cca cct cga taa         1338His Glu Arg Cys Asp Cys Ile Cys Ser Ser Ar #g Pro Pro Arg  *            360       #           365       #           370gagaatgtgc acatccttac attaagcctg aaagaacctt tagtttaagg ag#ggtgagat   1398aagagaccct tttcctacca gcaaccaaac ttactactag cctgcaatgc aa#tgaacaca   1458agtggttgct gagtctcagc cttgctttgt taatgccatg gcaagtagaa ag#gtatatca   1518tcaacttcta tacctaagaa tataggattg catttaataa tagtgtttga gg#ttatatat   1578gcacaaacac acacagaaat atattcatgt ctatgtgtat atagatcaaa tg#tttttttt   1638ttttggtata tataaccagg tacaccagag gttacatatg tttgagttag ac#tcttaaaa   1698tcctttgcca aaataaggga tggtcaaata tatgaaacat gtctttagaa aa#tttaggag   1758ataaatttat ttttaaattt tgaaacacga aacaattttg aatcttgctc tc#ttaaagaa   1818agcatcttgt atattaaaaa tcaaaagatg aggctttctt acatatacat ct#tagttgat   1878 tatt                  #                  #                   #           1882 <210> SEQ ID NO 37<211> LENGTH: 370 <212> TYPE: PRT <213> ORGANISM: Homo sapiens<400> SEQUENCE: 37 Met His Arg Leu Ile Phe Val Tyr Thr Leu Il#e Cys Ala Asn Phe Cys  1               5   #                10  #                15 Ser Cys Arg Asp Thr Ser Ala Thr Pro Gln Se#r Ala Ser Ile Lys Ala             20       #            25      #            30 Leu Arg Asn Ala Asn Leu Arg Arg Asp Glu Se#r Asn His Leu Thr Asp         35           #        40          #        45 Leu Tyr Arg Arg Asp Glu Thr Ile Gln Val Ly#s Gly Asn Gly Tyr Val     50               #    55              #    60 Gln Ser Pro Arg Phe Pro Asn Ser Tyr Pro Ar#g Asn Leu Leu Leu Thr 65                   #70                  #75                   #80 Trp Arg Leu His Ser Gln Glu Asn Thr Arg Il#e Gln Leu Val Phe Asp                 85   #                90  #                95 Asn Gln Phe Gly Leu Glu Glu Ala Glu Asn As#p Ile Cys Arg Tyr Asp             100       #           105      #           110 Phe Val Glu Val Glu Asp Ile Ser Glu Thr Se#r Thr Ile Ile Arg Gly         115           #       120          #       125 Arg Trp Cys Gly His Lys Glu Val Pro Pro Ar#g Ile Lys Ser Arg Thr     130               #   135              #   140 Asn Gln Ile Lys Ile Thr Phe Lys Ser Asp As#p Tyr Phe Val Ala Lys 145                 1 #50                 1#55                 1 #60 Pro Gly Phe Lys Ile Tyr Tyr Ser Leu Leu Gl#u Asp Phe Gln Pro Ala                 165   #               170  #               175 Ala Ala Ser Glu Thr Asn Trp Glu Ser Val Th#r Ser Ser Ile Ser Gly             180       #           185      #           190 Val Ser Tyr Asn Ser Pro Ser Val Thr Asp Pr#o Thr Leu Ile Ala Asp         195           #       200          #       205 Ala Leu Asp Lys Lys Ile Ala Glu Phe Asp Th#r Val Glu Asp Leu Leu     210               #   215              #   220 Lys Tyr Phe Asn Pro Glu Ser Trp Gln Glu As#p Leu Glu Asn Met Tyr 225                 2 #30                 2#35                 2 #40 Leu Asp Thr Pro Arg Tyr Arg Gly Arg Ser Ty#r His Asp Arg Lys Ser                 245   #               250  #               255 Lys Val Asp Leu Asp Arg Leu Asn Asp Asp Al#a Lys Arg Tyr Ser Cys             260       #           265      #           270 Thr Pro Arg Asn Tyr Ser Val Asn Ile Arg Gl#u Glu Leu Lys Leu Ala         275           #       280          #       285 Asn Val Val Phe Phe Pro Arg Cys Leu Leu Va#l Gln Arg Cys Gly Gly     290               #   295              #   300 Asn Cys Gly Cys Gly Thr Val Asn Trp Arg Se#r Cys Thr Cys Asn Ser 305                 3 #10                 3#15                 3 #20 Gly Lys Thr Val Lys Lys Tyr His Glu Val Le#u Gln Phe Glu Pro Gly                 325   #               330  #               335 His Ile Lys Arg Arg Gly Arg Ala Lys Thr Me#t Ala Leu Val Asp Ile             340       #           345      #           350 Gln Leu Asp His His Glu Arg Cys Asp Cys Il#e Cys Ser Ser Arg Pro         355           #       360          #       365 Pro Arg     370 <210> SEQ ID NO 38 <211> LENGTH: 20<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC21, #222<400> SEQUENCE: 38 tgagccctcg ccccagtcag             #                  #                   # 20 <210> SEQ ID NO 39 <211> LENGTH: 25<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC21, #224<400> SEQUENCE: 39 acatacagga aagccttgcc caaaa          #                   #               25 <210> SEQ ID NO 40<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: oligonucleotide primer ZC21,#223 <400> SEQUENCE: 40 aaactaccct gcgattctct gctgc          #                   #               25 <210> SEQ ID NO 41<211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: oligonucleotide primer ZC21,#334 <400> SEQUENCE: 41 ggtaaatgga gcttggctga g           #                   #                   #21 <210> SEQ ID NO 42<211> LENGTH: 3571 <212> TYPE: DNA <213> ORGANISM: Mus musculus<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1049)...(2086)<400> SEQUENCE: 42gaattcccgg gtcgacccac gcgtccgggc gcccagggga aaggaagctg gg#ggccgcct     60ggcggcattc ctcgccgcag tgtgggctcc gtctgccgcg gggcccgcag tg#ccccctgt    120ctgcgccagc acctgttggc ccgccagctg gccgcccgcg ccccccgcgc cc#cccgcgcc    180cgcccggccg ccagccccgc gccccgcgcg ccgcccgctg ggggaaagtg ga#gacgggga    240ggggacaaga gcgatcctcc aggccagcca ggccttccct tagccgcccg tg#cttagccg    300ccacctctcc tcagccctgc gtcctgccct gccttagggc aggcatccga gc#gctcgcga    360ctccgagccg cccaagctct cccggcttcc cgcagcactt cgccggtacc cg#agggaact    420tcggtggcca ccgactgcag caaggaggag gctccgcggt ggatccgggc ca#gtcccgag    480tcgtccccgc ggcctctctg cccgcccggg acccgcgcgg cactcgcagg gc#acggtccc    540ctccccccag gtgggggtgg ggcgccgcct gccgccccga tcagcagctt tg#tcattgat    600cccaaggtgc tcgcctcgct gccgacctgg cttccagtct ggcttggcgg ga#ccccgagt    660cctcgcctgt gtcctgtccc ccaaactgac aggtgctccc tgcgagtcgc ca#cgactcat    720cgccgctccc ccgcgtcccc accccttctt tcctccctcg cctaccccca cc#ccccgcac    780ttcggcacag ctcaggattt gtttaaacct tgggaaactg gttcaggtcc ag#gttttgct    840ttgatccttt tcaaaaactg gagacacaga agagggctct aggaaaaact tt#tggatggg    900attatgtgga aactaccctg cgattctctg ctgccagagc cggccaggcg ct#tccaccgc    960agcgcagcct ttccccggct gggctgagcc ttggagtcgt cgcttcccca gt#gcccgccg   1020 cgagtgagcc ctcgccccag tcagccaa atg ctc ctc ctc ggc #ctc ctc ctg      1072                    #             Met Leu Leu #Leu Gly Leu Leu Leu                    #              1    #           5 ctg aca tct gcc ctg gcc ggc caa aga acg gg#g act cgg gct gag tcc     1120Leu Thr Ser Ala Leu Ala Gly Gln Arg Thr Gl #y Thr Arg Ala Glu Ser     10              #     15              #     20aac ctg agc agc aag ttg cag ctc tcc agc ga#c aag gaa cag aac gga     1168Asn Leu Ser Ser Lys Leu Gln Leu Ser Ser As #p Lys Glu Gln Asn Gly 25                  # 30                  # 35                  # 40gtg caa gat ccc cgg cat gag aga gtt gtc ac#t ata tct ggt aat ggg     1216Val Gln Asp Pro Arg His Glu Arg Val Val Th #r Ile Ser Gly Asn Gly                 45  #                 50  #                 55agc atc cac agc ccg aag ttt cct cat aca ta#c cca aga aat atg gtg     1264Ser Ile His Ser Pro Lys Phe Pro His Thr Ty #r Pro Arg Asn Met Val             60      #             65      #             70ctg gtg tgg aga tta gtt gca gta gat gaa aa#t gtg cgg atc cag ctg     1312Leu Val Trp Arg Leu Val Ala Val Asp Glu As #n Val Arg Ile Gln Leu         75          #         80          #         85aca ttt gat gag aga ttt ggg ctg gaa gat cc#a gaa gac gat ata tgc     1360Thr Phe Asp Glu Arg Phe Gly Leu Glu Asp Pr #o Glu Asp Asp Ile Cys     90              #     95              #    100aag tat gat ttt gta gaa gtt gag gag ccc ag#t gat gga agt gtt tta     1408Lys Tyr Asp Phe Val Glu Val Glu Glu Pro Se #r Asp Gly Ser Val Leu105                 1 #10                 1 #15                 1 #20gga cgc tgg tgt ggt tct ggg act gtg cca gg#a aag cag act tct aaa     1456Gly Arg Trp Cys Gly Ser Gly Thr Val Pro Gl #y Lys Gln Thr Ser Lys                125   #               130   #               135gga aat cat atc agg ata aga ttt gta tct ga#t gag tat ttt cca tct     1504Gly Asn His Ile Arg Ile Arg Phe Val Ser As #p Glu Tyr Phe Pro Ser            140       #           145       #           150gaa ccc gga ttc tgc atc cac tac agt att at#c atg cca caa gtc aca     1552Glu Pro Gly Phe Cys Ile His Tyr Ser Ile Il #e Met Pro Gln Val Thr        155           #       160           #       165gaa acc acg agt cct tcg gtg ttg ccc cct tc#a tct ttg tca ttg gac     1600Glu Thr Thr Ser Pro Ser Val Leu Pro Pro Se #r Ser Leu Ser Leu Asp    170               #   175               #   180ctg ctc aac aat gct gtg act gcc ttc agt ac#c ttg gaa gag ctg att     1648Leu Leu Asn Asn Ala Val Thr Ala Phe Ser Th #r Leu Glu Glu Leu Ile185                 1 #90                 1 #95                 2 #00cgg tac cta gag cca gat cga tgg cag gtg ga#c ttg gac agc ctc tac     1696Arg Tyr Leu Glu Pro Asp Arg Trp Gln Val As #p Leu Asp Ser Leu Tyr                205   #               210   #               215aag cca aca tgg cag ctt ttg ggc aag gct tt#c ctg tat ggg aaa aaa     1744Lys Pro Thr Trp Gln Leu Leu Gly Lys Ala Ph #e Leu Tyr Gly Lys Lys            220       #           225       #           230agc aaa gtg gtg aat ctg aat ctc ctc aag ga#a gag gta aaa ctc tac     1792Ser Lys Val Val Asn Leu Asn Leu Leu Lys Gl #u Glu Val Lys Leu Tyr        235           #       240           #       245agc tgc aca ccc cgg aac ttc tca gtg tcc at#a cgg gaa gag cta aag     1840Ser Cys Thr Pro Arg Asn Phe Ser Val Ser Il #e Arg Glu Glu Leu Lys    250               #   255               #   260agg aca gat acc ata ttc tgg cca ggt tgt ct#c ctg gtc aag cgc tgt     1888Arg Thr Asp Thr Ile Phe Trp Pro Gly Cys Le #u Leu Val Lys Arg Cys265                 2 #70                 2 #75                 2 #80gga gga aat tgt gcc tgt tgt ctc cat aat tg#c aat gaa tgt cag tgt     1936Gly Gly Asn Cys Ala Cys Cys Leu His Asn Cy #s Asn Glu Cys Gln Cys                285   #               290   #               295gtc cca cgt aaa gtt aca aaa aag tac cat ga#g gtc ctt cag ttg aga     1984Val Pro Arg Lys Val Thr Lys Lys Tyr His Gl #u Val Leu Gln Leu Arg            300       #           305       #           310cca aaa act gga gtc aag gga ttg cat aag tc#a ctc act gat gtg gct     2032Pro Lys Thr Gly Val Lys Gly Leu His Lys Se #r Leu Thr Asp Val Ala        315           #       320           #       325ctg gaa cac cac gag gaa tgt gac tgt gtg tg#t aga gga aac gca gga     2080Leu Glu His His Glu Glu Cys Asp Cys Val Cy #s Arg Gly Asn Ala Gly    330               #   335               #   340ggg taa ctgcagcctt cgtagcagca cacgtgagca ctggcattct gt#gtaccccc      2136 Gly  * 345acaagcaacc ttcatcccca ccagcgttgg ccgcagggct ctcagctgct ga#tgctggct   2196atggtaaaga tcttactcgt ctccaaccaa attctcagtt gtttgcttca at#agccttcc   2256cctgcaggac ttcaagtgtc ttctaaaaga ccagaggcac caagaggagt ca#atcacaaa   2316gcactgcctt ctagaggaag cccagacaat ggtcttctga ccacagaaac aa#atgaaatg   2376aatgtagatc gctagcaaac tctggagtga cagcatttct tttccactga ca#gaatggtg   2436tagcttagtt gtcttgatat gggcaagtga tgtcagcaca agaaaatggt ga#aaaacaca   2496cacttgattg tgaacaatgc agaaatactt ggatttctcc aacctgtttg ca#tagataga   2556cagatgctct gttttctaca aactcaaagc ttttagagag cagctatgtt aa#taggaatt   2616aaatgtgcca tgctgaaagg aaagactgaa gttttcaatg cttggcaact tc#tccgcaat   2676ttggaggaaa ggtgcggtca tggtttggag aaagcacacc tgcacagagg ag#tggccttc   2736ccttcccttc cctctgaggt ggcttctgtg tttcattgtg tatattttta ta#ttctcctt   2796ttgacattat aactgttggc ttttctaatc ttgttaaata tttctatttt ta#ccaaaggt   2856atttaatatt cttttttatg acaacctaga gcaattattt ttagcttgat aa#tttttttt   2916tctaaacaaa attgttatag ccagaagaac aaagatgatt gatataaaaa tc#ttgttgct   2976ctgacaaaaa catatgtatt tcttccttgt atggtgctag agcttagcgt ca#tctgcatt   3036tgaaaagatg gaatggggaa gtttttagaa ttggtaggtc gcagggacag tt#tgataaca   3096actgtactat catcaattcc caattctgtt cttagagcta cgaacagaac ag#agcttgag   3156taaatatgga gccattgcta acctacccct ttctatggga aataggagta ta#gctcagag   3216aagcacgtcc ccagaaacct cgaccatttc taggcacagt gttctgggct at#gctgcgct   3276gtatggacat atcctattta tttcaatact agggttttat tacctttaaa ct#ctgctcca   3336tacacttgta ttaatacatg gatattttta tgtacagaag tatatcattt aa#ggagttca   3396cttattatac tctttggcaa ttgcaaagaa aatcaacata atacattgct tg#taaatgct   3456taatctgtgc ccaagttttg tggtgactat ttgaattaaa atgtattgaa tc#atcaaata   3516aaataatctg gctattttgg ggaaaaaaaa aaaaaaaaaa aaaaagggcg gc#cgc        3571 <210> SEQ ID NO 43 <211> LENGTH: 345 <212> TYPE: PRT<213> ORGANISM: Mus musculus <400> SEQUENCE: 43Met Leu Leu Leu Gly Leu Leu Leu Leu Thr Se #r Ala Leu Ala Gly Gln 1               5   #                10   #                15Arg Thr Gly Thr Arg Ala Glu Ser Asn Leu Se #r Ser Lys Leu Gln Leu            20       #            25       #            30Ser Ser Asp Lys Glu Gln Asn Gly Val Gln As #p Pro Arg His Glu Arg        35           #        40           #        45Val Val Thr Ile Ser Gly Asn Gly Ser Ile Hi #s Ser Pro Lys Phe Pro    50               #    55               #    60His Thr Tyr Pro Arg Asn Met Val Leu Val Tr #p Arg Leu Val Ala Val65                   #70                   #75                   #80Asp Glu Asn Val Arg Ile Gln Leu Thr Phe As #p Glu Arg Phe Gly Leu                85   #                90   #                95Glu Asp Pro Glu Asp Asp Ile Cys Lys Tyr As #p Phe Val Glu Val Glu            100       #           105       #           110Glu Pro Ser Asp Gly Ser Val Leu Gly Arg Tr #p Cys Gly Ser Gly Thr        115           #       120           #       125Val Pro Gly Lys Gln Thr Ser Lys Gly Asn Hi #s Ile Arg Ile Arg Phe    130               #   135               #   140Val Ser Asp Glu Tyr Phe Pro Ser Glu Pro Gl #y Phe Cys Ile His Tyr145                 1 #50                 1 #55                 1 #60Ser Ile Ile Met Pro Gln Val Thr Glu Thr Th #r Ser Pro Ser Val Leu                165   #               170   #               175Pro Pro Ser Ser Leu Ser Leu Asp Leu Leu As #n Asn Ala Val Thr Ala            180       #           185       #           190Phe Ser Thr Leu Glu Glu Leu Ile Arg Tyr Le #u Glu Pro Asp Arg Trp        195           #       200           #       205Gln Val Asp Leu Asp Ser Leu Tyr Lys Pro Th #r Trp Gln Leu Leu Gly    210               #   215               #   220Lys Ala Phe Leu Tyr Gly Lys Lys Ser Lys Va #l Val Asn Leu Asn Leu225                 2 #30                 2 #35                 2 #40Leu Lys Glu Glu Val Lys Leu Tyr Ser Cys Th #r Pro Arg Asn Phe Ser                245   #               250   #               255Val Ser Ile Arg Glu Glu Leu Lys Arg Thr As #p Thr Ile Phe Trp Pro            260       #           265       #           270Gly Cys Leu Leu Val Lys Arg Cys Gly Gly As #n Cys Ala Cys Cys Leu        275           #       280           #       285His Asn Cys Asn Glu Cys Gln Cys Val Pro Ar #g Lys Val Thr Lys Lys    290               #   295               #   300Tyr His Glu Val Leu Gln Leu Arg Pro Lys Th #r Gly Val Lys Gly Leu305                 3 #10                 3 #15                 3 #20His Lys Ser Leu Thr Asp Val Ala Leu Glu Hi #s His Glu Glu Cys Asp                325   #               330   #               335Cys Val Cys Arg Gly Asn Ala Gly Gly             340      #           345 <210> SEQ ID NO 44 <211> LENGTH: 65 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC20, #572<400> SEQUENCE: 44tcaccacgcg aattcggtac cgctggttcc gcgtggatcc ggccagagac ag#gggactca     60 ggcgg                  #                  #                   #            65 <210> SEQ ID NO 45 <211> LENGTH: 65<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC20, #573<400> SEQUENCE: 45tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ctatcctcct gt#gctccctc     60 tgcac                  #                  #                   #            65 <210> SEQ ID NO 46 <211> LENGTH: 40<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC19, #372<400> SEQUENCE: 46 tgtcgatgaa gccctgaaag acgcgcagac taattcgagc     #                   #    40 <210> SEQ ID NO 47 <211> LENGTH: 60<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC19, #351<400> SEQUENCE: 47acgcgcagac taattcgagc tcccaccatc accatcacca cgcgaattcg gt#accgctgg     60 <210> SEQ ID NO 48 <211> LENGTH: 60 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC19, #352<400> SEQUENCE: 48actcactata gggcgaattg cccgggggat ccacgcggaa ccagcggtac cg#aattcgcg     60 <210> SEQ ID NO 49 <211> LENGTH: 42 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: oligonucleotide primer ZC19, #371<400> SEQUENCE: 49 acggccagtg aattgtaata cgactcacta tagggcgaat tg    #                   #  42 <210> SEQ ID NO 50 <211> LENGTH: 1095<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Fused DNA <400> SEQUENCE: 50ctgaaagacg cgcagactaa ttcgagctcc caccatcacc atcaccacgc ga#attcggta     60ccgctggttc cgcgtggatc cggccagaga caggggactc aggcggaatc ca#acctgagt    120agtaaattcc agttttccag caacaaggaa cagaacggag tacaagatcc tc#agcatgag    180agaattatta ctgtgtctac taatggaagt attcacagcc caaggtttcc tc#atacttat    240ccaagaaata cggtcttggt atggagatta gtagcagtag aggaaaatgt at#ggatacaa    300cttacgtttg atgaaagatt tgggcttgaa gacccagaag atgacatatg ca#agtatgat    360tttgtagaag ttgaggaacc cagtgatgga actatattag ggcgctggtg tg#gttctggt    420actgtaccag gaaaacagat ttctaaagga aatcaaatta ggataagatt tg#tatctgat    480gaatattttc cttctgaacc agggttctgc atccactaca acattgtcat gc#cacaattc    540acagaagctg tgagtccttc agtgctaccc ccttcagctt tgccactgga cc#tgcttaat    600aatgctataa ctgcctttag taccttggaa gaccttattc gatatcttga ac#cagagaga    660tggcagttgg acttagaaga tctatatagg ccaacttggc aacttcttgg ca#aggctttt    720gtttttggaa gaaaatccag agtggtggat ctgaaccttc taacagagga gg#taagatta    780tacagctgca cacctcgtaa cttctcagtg tccataaggg aagaactaaa ga#gaaccgat    840accattttct ggccaggttg tctcctggtt aaacgctgtg gtgggaactg tg#cctgttgt    900ctccacaatt gcaatgaatg tcaatgtgtc ccaagcaaag ttactaaaaa at#accacgag    960gtccttcagt tgagaccaaa gaccggtgtc aggggattgc acaaatcact ca#ccgacgtg   1020gccctggagc accatgagga gtgtgactgt gtgtgcagag ggagcacagg ag#gatagtgt   1080 tttggcggat gagat               #                  #                   #  1095

We claim:
 1. A method of decreasing zvegf3 activity in a mammal,comprising administering to the mammal an effective amount of anantibody that specifically binds to an epitope of a polypeptideconsisting of a sequence of amino acid residues selected from the groupconsisting of: residues 230-345 of SEQ ID NO:2; residues 231-345 of SEQID NO:2; residues 232-345 of SEQ ID NO:2; residues 233-345 of SEQ IDNO:2; residues 234-345 of SEQ ID NO:2; residues 235-345 of SEQ ID NO:2;residues 236-345 of SEQ ID NO:2; residues 237-345 of SEQ ID NO:2;residues 238-345 of SEQ ID NO:2; residues 239-345 of SEQ ID NO:2; andresidues 240-345 of SEQ ID NO:2.
 2. The method of claim 1 wherein theantibody is a monoclonal antibody.
 3. The method of claim 2 wherein theantibody is a humanized antibody.
 4. The method of claim 1 wherein theantibody is a single-chain antibody.
 5. A method of decreasing zvegf3activity in a mammal, comprising administering to the mammal aneffective amount of an antibody that specifically binds to a dimericprotein having two polypeptide chains, wherein each of said polypeptidechains consists of a sequence of amino acid residues selected from thegroup consisting of: residues 230-345 of SEQ ID NO:2; residues 231-345of SEQ ID NO:2; residues 232-345 of SEQ ID NO:2; residues 233-345 of SEQID NO:2; residues 234-345 of SEQ ID NO:2; residues 235-345 of SEQ IDNO:2; residues 236-345 of SEQ ID NO:2; residues 237-345 of SEQ ID NO:2;residues 238-345 of SEQ ID NO:2; residues 239-345 of SEQ ID NO:2; andresidues 240-345 of SEQ ID NO:2.
 6. The method of claim 5 wherein theantibody is a monoclonal antibody.
 7. The method of claim 6 wherein theantibody is a humanized antibody.
 8. The method of claim 5 wherein theantibody is a single-chain antibody.